Methods and uses for treating cancer using antibody-drug conjugates

Abstract

Provided herein are methods related to treating or delaying progression of cancer and / or predicting outcome of treating an individual having cancer, as well as uses, systems, and non-transitory computer readable storage media related thereto. In some embodiments, the methods comprise acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual and administering to the individual a treatment comprising an antibody-drug conjugate (ADC) that targets the antigen.

Description

Attorney Docket No: 197102019240METHODS AND USES FOR TREATING CANCER USING ANTIBODY-DRUG CONJUGATESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 688,732, filed August 29, 2024, which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] Provided herein are methods related to treating or delaying progression of cancer and / or predicting outcome of treating an individual having cancer, as well as uses, systems, and non- transitory computer readable storage media related thereto. In some embodiments, the methods comprise acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual and administering to the individual a treatment comprising an antibody-drug conjugate (ADC) that targets the antigen.BACKGROUND

[0003] Antibody-drug conjugates (ADCs) have become a powerful tool for treating various types of cancers. Generally, these comprise an antibody that specifically binds a cancer-expressed antigen fused to a cytotoxic pay load (e.g., a cytotoxic agent or moiety) via linker. This allows the targeting of highly potent cytotoxic payloads directly to cancer cells via specific antigen recognition.

[0004] One cancer-expressed antigen for which multiple ADCs have been developed is HER2 (see, e.g., Rassy, E. et al. (2022) Breast 66:217-226). HER2-targeted therapy is broadly used in advanced breast cancer (aBC). For patients whose cancers express HER2, first-line standard of care treatment includes unconjugated HER2 antibodies trastuzumab and pertuzumab (HP) in combination with chemotherapy. However, ADCs targeting HER2 are used for second-line or higher (2L+) treatment of HER2-low cancers as well as adjuvant treatment of patients with HER2-positive early breast cancer who have residual invasive disease after neoadjuvant taxane-based chemotherapy and trastuzumabbased treatment (see Wedam, S. et al. (2020) Clin Cancer Res. 26(16):4180-4185). ADCs targeting cancer antigens other than HER2 have also been approved for cancer treatment, including those targeting CD19, CD22, CD30, CD33, CD79b, c-MET, Nectin-4, Trop-2, Tissue Factor (TF), claudin 18, and folate receptor a (see Liu, K. et al. (2024) Mol Cancer. 23( 1) :62).

[0005] Even though ADCs represent a powerful class of cancer therapeutic, not all patients respond to treatment. Thus, there is a need in the art for identifying those patients likely to respond to ADCbased treatment and those that are not, as well as for predicting treatment outcomes to ADC-based therapies.1MOFO-359650473Attorney Docket No: 197102019240

[0006] All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control.SUMMARY OF THE INVENTION

[0007] In one aspect, provided herein is a method of treating or delaying progression of cancer in an individual, comprising: acquiring knowledge of or detecting a copy number (CN) ratio of greater than or equal to 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and administering to the individual a treatment comprising an antibody-drug conjugate (ADC), wherein the antibody of the ADC specifically binds the antigen.

[0008] In another aspect, provided herein is a method of predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the antibody of the ADC specifically binds the antigen, wherein the CN ratio of the antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC. In another aspect, provided herein is a method of predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the antibody of the ADC specifically binds the antigen, wherein the CN ratio of the antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample, and wherein a CN ratio of greater than 0.5 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than or equal to 0.5 with a treatment comprising the ADC. In some embodiments, the improved outcome comprises longer progression-free survival (PFS) (e.g., real world PFS, rwPFS), longer overall survival (OS) (e.g., real world OS, rwOS), and / or longer time to treatment discontinuation (TTD).

[0009] In some embodiments according to any one of the embodiments disclosed herein, the CN ratio of the gene encoding the antigen in the sample is greater than 2.5 (and optionally less than or equal to 5). In some embodiments, the CN ratio of the gene encoding the antigen in the sample is2MOFO-359650473Attorney Docket No: 197102019240 greater than 5 (and optionally less than or equal to 10). In some embodiments, the CN ratio of the gene encoding the antigen in the sample is greater than 10, greater than 15, or greater than 20.

[0010] In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: acquiring knowledge of or detecting a copy number (CN) ratio of less than 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from an individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and administering to the individual a therapeutic agent other than an antibody-drug conjugate (ADC). In another aspect, provided herein is a method of treating or delaying progression of cancer, comprising: acquiring knowledge of or detecting a copy number (CN) ratio of less than or equal to 0.5 of a gene encoding an antigen expressed by the cancer in a sample obtained from an individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and administering to the individual a therapeutic agent other than an antibody-drug conjugate (ADC). In some embodiments, the therapeutic agent other than an ADC comprises a standard of care treatment or a chemotherapeutic agent.

[0011] In some embodiments according to any one of the embodiments disclosed herein, the ploidy of the sample refers to an average ploidy of two or more segments of the genome. In some embodiments, ploidy of the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), comparative genomic hybridization (CGH), or microarray analysis. In some embodiments, CN of the gene encoding the antigen in the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), in situ hybridization (ISH), comparative genomic hybridization (CGH), or microarray analysis. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

[0012] In some embodiments according to any one of the embodiments disclosed herein, the methods further comprise obtaining the sample from the individual. In some embodiments, the sample is obtained from the cancer. In some embodiments, the sample comprises a tissue biopsy sample or a liquid biopsy sample. In some embodiments, the sample is from a tumor biopsy or tumor specimen. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises DNA, circulating tumor DNA (ctDNA), or cell- free DNA from the cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is biliary tract cancer, breast cancer, gastric cancer, colorectal cancer, or lung cancer. In some embodiments, the cancer is advanced or metastatic.3MOFO-359650473Attorney Docket No: 197102019240

[0013] In some embodiments according to any one of the embodiments disclosed herein, the treatment comprising the ADC is a first-line (IL) treatment. In some embodiments, the treatment comprising the ADC is an adjuvant or neoadjuvant treatment. In some embodiments, the treatment comprising the ADC is a second-line (2L) or higher treatment. In some embodiments, the antigen expressed by the cancer is expressed on the surface of cells of the cancer. In some embodiments, the antigen expressed by the cancer is HER2, and the antibody of the ADC specifically binds HER2. In some embodiments, the ADC is trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), disitamab vedotin (RC48), trastuzumab duocarmazine (SYD985), XMT-1522, ZRC-3256, MRG002, ARX788, BDC-1001, A166, FS-1502, SHR-A1201, DP303c, BI-CON-02, ALT-P7, DX126-262, ZW49, HS630, B003, SBT6050, SHR-A1811, MT-5111, GQ1001, PF-06804103, or T-PNU. In some embodiments, the cancer expresses HER2 at a level of 3 or higher, as measured by immunohistochemistry (IHC), or wherein the cancer is positive for HER2 expression by in situ hybridization (ISH) and shows HER2 expression at a level of 2 or higher as measured by IHC. In some embodiments, the cancer expresses HER2 at a level of 0 as measured by IHC, or wherein the cancer is negative for HER2 expression by ISH and shows HER2 expression at a level of 1 or higher or 2 or higher. In some embodiments, the individual has received prior treatment with a HER2- targeted therapy. In some embodiments, the HER2-targeted therapy comprises trastuzumab and / or pertuzumab. In some embodiments, the individual has not received prior treatment with a HER2- targeted therapy. In some embodiments, the antigen expressed by the cancer is c-mesenchymal- epithelial transition (c-MET), and the antibody of the ADC specifically binds c-MET. In some embodiments, the ADC is telisotuzumab vedotin (Teliso-V, ABBV-399), RC108, or HRA00129- C004. In some embodiments, the antigen expressed by the cancer is trophoblast cell surface antigen 2 (Trop-2), and the antibody of the ADC specifically binds Trop-2. In some embodiments, the ADC is sacituzumab govitecan, datopotamab deruxtecan (Dato-DXd), SKB-264, BIO-106, DB-1305, ESG- 401, BAT-8008, BL-M02D1, DAC-002 (JS108), FDA018, or SHR-A1921. In some embodiments, the antigen expressed by the cancer is folate receptor alpha (FRa), and the antibody of the ADC specifically binds FRa. In some embodiments, the ADC is mirvetuximab soravtansine, luveltamab tazevibulin (STRG-002), or MORAb-202. In some embodiments, the antigen expressed by the cancer is claudin 18, and the antibody of the ADC specifically binds claudin 18. In some embodiments, the ADC is CMG901, IBI343, SKB315 (MK-1200), TORL-2-307-ADC, SYSA1801, AZD0901 (CMG901), ATG-022, SOT102, or EO-3021.

[0014] In another aspect, provided herein is a system for predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to perform a method according to any one of the above embodiments. In some embodiments, the one or more program instructions when executed by the one4MOFO-359650473Attorney Docket No: 197102019240 or more processors are configured to: obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; analyze the plurality of sequence reads for copy number of a gene encoding an antigen expressed by the cancer; and calculate, based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples, wherein the CN ratio refers to CN of the gene encoding the antigen in the one or more samples divided by ploidy of the one or more samples; wherein the antibody of the ADC specifically binds the antigen, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.

[0015] In another aspect, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method according to any one of the above embodiments. In some embodiments, the non-transitory computer readable storage medium comprises one or more programs executable by one or more computer processors for predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), the method comprising: obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; analyzing, using the one or more processors, the plurality of sequence reads for copy number of a gene encoding an antigen expressed by the cancer; and calculating, using the one or more processors and based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples, wherein the CN ratio refers to CN of the gene encoding the antigen in the one or more samples divided by ploidy of the one or more samples; wherein the antibody of the ADC specifically binds the antigen, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.

[0016] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 illustrates the schema and cohorts used for a study on advanced breast cancer patients treated with HER2 antibody therapies to analyze whether HER2 genomic copy number ratio predicts treatment outcomes.5MOFO-359650473Attorney Docket No: 197102019240

[0018] FIGS. 2A-2C show time to treatment discontinuation (TTD; FIG. 2A), real-world progression-free survival (rwPFS; FIG. 2B), and real-world overall survival (rwOS; FIG. 2C) among HER2+ (HER2 IHC 3+ or 2+ / ISH+) advanced breast cancer (aBC) patients treated with trastuzumab and pertuzumab (HP) in combination with chemotherapy in first-line (IL; Cohort 1 in FIG. 1), comparing patients with an ERBB2 copy number (CN) ratio of >5 vs. <5.

[0019] FIGS. 3A-3C show TTD (FIG. 3A), rwPFS (FIG. 3B), and rwOS (FIG. 3C) among HER2+ (HER2 IHC 3+ or 2+ / ISH+) advanced breast cancer (aBC) patients treated with trastuzumab and pertuzumab (HP) in combination with chemotherapy in IL (Cohort 1 in FIG. 1), comparing patients with an ERBB2 copy number ratio of <2.5, 2.5-5, or >5, as indicated.

[0020] FIGS. 4A-4C show hazard ratios for TTD (FIG. 4A), rwPFS (FIG. 4B), and rwOS (FIG. 4C) among HER2+ (HER2 IHC 3+ or 2+ / ISH+) advanced breast cancer (aBC) patients treated with trastuzumab and pertuzumab (HP) in combination with chemotherapy in IL (Cohort 1 in FIG. 1), stratified by CN ratio.

[0021] FIGS. 5A-5C show TTD (FIG. 5A), rwPFS (FIG. 5B), and rwOS (FIG. 5C) among HER2+ (HER2 IHC 3+ or 2+ / ISH+) advanced breast cancer (aBC) patients treated with trastuzumab and pertuzumab (HP) in combination with chemotherapy in IE (Cohort 1 in FIG. 1), comparing patients with an ERBB2 CN ratio of <2.5, greater than 2.5 but less than or equal to 5, or >5, as indicated.

[0022] FIGS. 6A-6C show TTD (FIG. 6A), rwPFS (FIG. 6B), and rwOS (FIG. 6C) among HER2+ (HER2 IHC 3+ or 2+ / ISH+) advanced breast cancer (aBC) patients who have not received prior HER2-targeted therapies and who received treatment with trastuzumab deruxtecan (T-DXd) in the 2E or 3E setting (Cohort 2 in FIG. 1), comparing patients with an ERBB2 CN ratio of <2.5, greater than 2.5 but less than or equal to 5, or >5.

[0023] FIGS. 7A-7C show TTD (FIG. 7A), rwPFS (FIG. 7B), and rwOS (FIG. 7C) among HER2 low / negative (HER2 IHC 0 or 1+ to 2+ / ISH-) advanced breast cancer (aBC) patients who have not received prior HER2-targeted therapies and who received treatment with T-DXd in the 2E or 3E setting (Cohort 3 in FIG. 1), comparing patients with an ERBB2 CN ratio of <2.5 or greater than 2.5 but less than or equal to 5.

[0024] FIGS. 8A-8C show ERBB2 CN ratio (values shown as >x) vs. hazard ratio for TTD (FIG. 8A), rwPFS (FIG. 8B), and rwOS (FIG. 8C).

[0025] FIGS. 9A-9C show TTD (FIG. 9A), rwPFS (FIG. 9B), and rwOS (FIG. 9C) among HER2 low / negative (HER2 IHC 0 or 1+ to 2+ / ISH-) advanced breast cancer (aBC) patients who have not received prior HER2-targeted therapies and who received treatment with T-DXd in the 2E or 3E setting (Cohort 3 in FIG. 1), comparing patients with an ERBB2 CN ratio of greater than 0.5 vs. CN ratio less than or equal to 0.5.

[0026] FIG. 10 depicts an exemplary device, in accordance with some embodiments.

[0027] FIG. 11 depicts an exemplary system, in accordance with some embodiments.6MOFO-359650473Attorney Docket No: 197102019240

[0028] FIG. 12 depicts a block diagram of an exemplary process for predicting outcome to treatment with an ADC, in accordance with some embodiments.DETAILED DESCRIPTION

[0029] The present disclosure relates generally to detecting copy number (CN) ratio of a gene encoding an antigen in a sample, and its use in treating or delaying progression of cancer in an individual using an ADC or predicting outcome of treating an individual having cancer with an ADC. In particular, the present disclosure demonstrates that CN ratio of a gene encoding an antigen in a sample from an individual was significantly associated with clinical outcome of treating an individual with an ADC targeting the antigen, e.g., HER2. Benefit of ADC treatment was seen at a range of CN ratio values above a threshold CN ratio. Even low values of CN ratio were associated with benefit, though importantly not all non-zero values of CN ratio were associated with benefit. As such, CN ratio of a gene encoding antigen in a sample can be used, e.g., to stratify individuals for treatment comprising an ADC targeting the antigen and / or predict response of an individual to a treatment comprising an ADC targeting the antigen.

[0030] In some embodiments, provided herein are methods of treating or delaying progression of cancer in an individual, comprising acquiring knowledge of or detecting a copy number (CN) ratio of greater than or equal to 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and administering to the individual a treatment comprising an antibody-drug conjugate (ADC), wherein the antibody of the ADC specifically binds the antigen.

[0031] In some embodiments, provided herein are methods of predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the antibody of the ADC specifically binds the antigen, wherein the CN ratio of the antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.

[0032] In some embodiments, provided herein are methods of treating or delaying progression of cancer, comprising: acquiring knowledge of or detecting a copy number (CN) ratio of less than 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from an individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and administering to the individual a therapeutic agent other than an antibody-drug conjugate (ADC).7MOFO-359650473Attorney Docket No: 197102019240I. General Techniques

[0033] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).II. Definitions

[0034] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0035] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0036] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0037] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.8MOFO-359650473Attorney Docket No: 197102019240

[0038] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as referred to herein.

[0039] “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and doublestranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs.

[0040] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl-, 2'-fluoro-, or 2'-azido-ribose,9MOFO-359650473Attorney Docket No: 197102019240 carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), "(0)NR2 ("amidate"), P(0)R, P(0)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. A polynucleotide can contain one or more different types of modifications as described herein and / or multiple modifications of the same type. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0041] “Oligonucleotide,” as used herein, generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0042] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0043] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic, and / or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0044] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end10MOFO-359650473Attorney Docket No: 197102019240(VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0045] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“I”), based on the amino acid sequences of their constant domains.

[0046] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0047] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0048] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigenbinding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0049] The term “hypervariable region,” “HVR,” or “HV,” as used herein, refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1 -25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a11MOFO-359650473Attorney Docket No: 197102019240 heavy chain only are functional and stable in the absence of light chain. See, for example, Hamers- Casterman et al., Nature 363:446-448 (1 993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0050] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1 991 )). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.Loop Kabat AbM Chothia ContactLI L24-L34 L24-L34 E26-E32 L30-L36L2 L50-L56 L50-L56 E50-E52 L46-L55L3 L89-L97 L89-L97 E91-E96 L89-L96Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0051] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95- 102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0052] ‘ ‘Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

[0053] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0054] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 -107 of the light chain and residues 1 -1 13 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National12MOFO-359650473Attorney Docket No: 197102019240Institutes of Health, Bethesda, Md. (1991 )). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.

[0055] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[0056] “Antibody fragments” comprise a portion of an intact antibody comprising the antigenbinding region thereof. In some embodiments, the antibody fragment described herein is an antigenbinding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0057] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target-binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target-binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins .

[0058] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al.,13MOFO-359650473Attorney Docket No: 197102019240Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991 ); Marks et al., J. Mol. Biol. 222: 581 -597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-31 0 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1 -2): 1 1 9-132 (2004)), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741 ; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1 993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Eonberg et al., Intern. Rev. Immunol. 13: 65-93 (1995)).

[0059] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0060] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0061] A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0062] A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. For example, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0063] As used herein, the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target14MOFO-359650473Attorney Docket No: 197102019240(which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0064] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0065] The term “detection” includes any means of detecting, including direct and indirect detection. The term “biomarker” as used herein (e.g., a “biomarker” such as a mutant nucleic acid molecule or polypeptide described herein) refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and / or clinical features (e.g., responsiveness to therapy including a checkpoint inhibitor). In some embodiments, a biomarker is a collection of genes or a collective number of mutations / alterations (e.g., somatic mutations) in a collection of genes. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide alterations (e.g., polynucleotide copy number alterations, e.g., DNA copy number alterations), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0066] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.15MOFO-359650473Attorney Docket No: 197102019240

[0067] The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid.

[0068] The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, for instance, by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0069] The term “aiding diagnosis” is used herein to refer to methods that assist in making a clinical determination regarding the presence, or nature, of a particular type of symptom or condition of a disease or disorder (e.g., cancer). For example, a method of aiding diagnosis of a disease or condition (e.g., cancer) can comprise measuring certain somatic mutations in a biological sample from an individual.

[0070] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some instances, the sample is a16MOFO-359650473Attorney Docket No: 197102019240 whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is from a tumor (e.g., a “tumor sample”), such as from a biopsy. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0071] A “tumor cell” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0072] A “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refer to a sample, cell, tissue, standard, or level that is used for comparison purposes.

[0073] By “correlate” or “correlating” is meant comparing, in any way, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocol and / or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

[0074] ‘ ‘Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down or complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down, or complete stopping) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e. reduction, slowing down, or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extension in the length of survival, including overall survival and progression free survival; and / or (7) decreased mortality at a given point of time following treatment.

[0075] An “effective response” of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and / or progression-free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.

[0076] An “effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e.,17MOFO-359650473Attorney Docket No: 197102019240 slow to some extent and in some embodiments stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in some embodiments stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), response rates (e.g., CR and PR), duration of response, and / or quality of life.

[0077] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0078] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0079] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0080] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. In particular embodiments, the patient herein is a human.

[0081] As used herein, “administering” is meant a method of giving a dosage of an agent or a pharmaceutical composition (e.g., a pharmaceutical composition including the agent) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various timepoints, bolus administration, and pulse infusion are contemplated herein.

[0082] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent18MOFO-359650473Attorney Docket No: 197102019240 administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0083] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0084] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a reagent for specifically detecting a biomarker (e.g., a kinase fusion or a fusion nucleic acid molecule or polypeptide described herein) described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0085] The phrase “based on” when used herein means that the information about one or more biomarkers (e.g., a kinase fusion or a fusion nucleic acid molecule or polypeptide described herein) is used to inform a treatment decision, information provided on a package insert, or marketing / promotional guidance, etc.III. Methods, Systems, and Devices

[0086] Certain aspects of the present disclosure relate to methods for treating or delaying progression of cancer in an individual. Other aspects of the present disclosure relate to predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC). In some embodiments of any of the methods provided herein, the methods comprise acquiring knowledge of a copy number (CN) ratio of greater than or equal to 0.5, greater than 0.5, greater than or equal to 1, greater than 1, greater than or equal to 2.5, greater than 2.5, greater than or equal to 5, greater than 5, greater than or equal to 10, greater than 10, greater than or equal to 12, greater than 12, greater than or equal to 14, greater than 14, greater than or equal to 15, greater than 15, greater than or equal to 16, greater than 16, greater than or equal to 18, greater than 18, greater than or equal to 20, or greater than 20 of a gene encoding an antigen expressed by a cancer (e.g., a cancer-expressed antigen) in a sample obtained from an individual. In some embodiments of any of the methods provided herein, the methods comprise detecting a copy number (CN) ratio of greater than or equal to 0.5, greater than 0.5, greater than or equal to 1, greater than 1, greater than or equal to 2.5, greater than 2.5, greater than or equal to 5, greater than 5, greater than or equal to 10, greater than 10, greater than or equal to 12, greater than 12, greater than or equal to 14, greater than 14, greater than or equal to 15, greater than 15, greater than or equal to 16, greater than 16, greater than or equal to 18, greater than 18, greater than or equal to 20, or greater than 20 of a gene encoding an antigen expressed by a cancer (e.g., a cancer-expressed antigen) in a sample obtained from an individual. In some embodiments, the19MOFO-359650473Attorney Docket No: 197102019240CN ratio is greater than or equal to 0.5, greater than or equal to 1, greater than or equal to 2, greater than or equal to 2.5, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 7.5, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, greater than or equal to 11, greater than or equal to 12, greater than or equal to 13, greater than or equal to 14, greater than or equal to 15, greater than or equal to 16, greater than or equal to 17, greater than or equal to 18, greater than or equal to 19, or greater than or equal to 20. In some embodiments, the CN ratio is less than or equal to 5, less than or equal to 6, less than or equal to 7, less than or equal to 8, less than or equal to 9, or less than or equal to 10. In some embodiments, CN ratio of the gene encoding antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample. In some embodiments, a CN ratio of greater than or equal to 1 of the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising an ADC whose antibody component specifically binds the antigen, as compared to outcome of treating an individual whose sample has a CN ratio of the antigen that is less than 1 with a treatment comprising the ADC. In some embodiments, a CN ratio of greater than or equal to 0.5 of the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising an ADC whose antibody component specifically binds the antigen, as compared to outcome of treating an individual whose sample has a CN ratio of the antigen that is less than 0.5 with a treatment comprising the ADC. In some embodiments, the methods further comprise administering to the individual a treatment comprising an ADC whose antibody component specifically binds the antigen, e.g., in an effective amount.

[0087] In some embodiments, the methods provided herein comprise acquiring knowledge of a copy number (CN) ratio of less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5 (and optionally greater than 0) of a gene encoding an antigen expressed by a cancer in a sample obtained from an individual. In some embodiments, the methods provided herein comprise detecting a copy number (CN) ratio of less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5 (and optionally greater than 0) of a gene encoding an antigen expressed by a cancer in a sample obtained from an individual. In some embodiments, a CN ratio of less than 1 of the gene encoding the antigen in the sample predicts a worse outcome associated with treating the individual with a treatment comprising an ADC whose antibody component specifically binds the antigen, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is greater than or equal to 1 with a treatment comprising the ADC. In some embodiments, the methods further comprise administering to the individual a treatment comprising a therapeutic agent other than an ADC, e.g., in an effective amount.

[0088] Other aspects of the present disclosure relate to systems for performing any of the methods disclosed herein, e.g., for predicting outcome of treating an individual having cancer with a treatment comprising an ADC. In some embodiments, the systems comprise a memory configured to store one20MOFO-359650473Attorney Docket No: 197102019240 or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; analyze the plurality of sequence reads for copy number of an antigen expressed by the cancer; and calculate, based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples.

[0089] Other aspects of the present disclosure relate to non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing any of the methods disclosed herein, e.g., for predicting outcome of treating an individual having cancer with a treatment comprising an ADC. In some embodiments, the methods comprise obtaining, using one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer; analyzing, using the one or more processors, the plurality of sequence reads for copy number of an antigen expressed by the cancer; and calculating, using the one or more processors and based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples.Copy number ratio

[0090] Certain aspects of the present disclosure relate to copy number (CN) ratio of a gene encoding an antigen in a sample. In some embodiments, CN ratio of a gene encoding an antigen in a sample refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample.

[0091] A variety of CNs (e.g., of a gene encoding an antigen of the present disclosure) may be used or selected by the skilled person guided by the present disclosure. In some embodiments, a CN of greater than or equal to 1 , greater than 1 , greater than or equal to 2, greater than 2, greater than or equal to 5, greater than 5, greater than or equal to 10, greater than 10, greater than or equal to 20, greater than 20, greater than or equal to 24, greater than 24, greater than or equal to 28, greater than 28, greater than or equal to 30, greater than 30, greater than or equal to 32, greater than 32, greater than or equal to 36, greater than 36, greater than or equal to 40, or greater than 40 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising an ADC of the present disclosure e.g., whose antibody component specifically binds the antigen). In some embodiments, the CN is less than or equal to 10, less than or equal to 12, less than or equal to 14, less than or equal to 16, less than or equal to 18, or less than or equal to 20. In some embodiments, a CN of a gene encoding an antigen that predicts improved outcome to treatment with an ADC targeting the antigen (e.g., whose antibody component specifically binds the antigen) is any CN having an upper limit of less than or equal to 5, 6, 8, 10, 12, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, or 80 and an independently selected lower limit of21MOFO-359650473Attorney Docket No: 197102019240 greater than or equal to 1, 2, 5, 6, 8, 10, 12, 15, 16, 18, or 20, wherein the upper limit is greater than the lower limit.

[0092] Techniques for assaying CN of a gene encoding an antigen are known in the art. In some embodiments, CN is determined by sequencing, e.g., massively parallel sequencing (MPS), whole genome sequencing (WGS), or next-generation sequencing (NGS). In some embodiments, CN is determined by real-time polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), or droplet digital PCR (ddPCR). In some embodiments, CN is determined by in situ hybridization (ISH), comparative genomic hybridization (CGH), or microarray analysis.

[0093] In array-based CGH methods, a first sample of nucleic acids (e.g., from a sample, such as from a tumor, or a tissue or liquid biopsy) is labeled with a first label, while a second sample of nucleic acids e.g., a control, such as from a healthy cell / tissue) is labeled with a second label. In some embodiments, equal quantities of the two samples are mixed and co-hybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments or oligonucleotides, which have been spotted in triplicate on the array. After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. The resulting ratio of the fluorescence intensities is proportional to the ratio of the copy numbers of DNA sequences in the two samples. In some embodiments, where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels are detected and the ratio provides a measure of the copy number. Array-based CGH can also be performed with single-color labeling. In single color CGH, a control (e.g., control nucleic acid sample, such as from a healthy cell / tissue) is labeled and hybridized to one array and absolute signals are read, and a test sample (e.g., a nucleic acid sample obtained from an individual or from a tumor, or a tissue or liquid biopsy) is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number differences are calculated based on absolute signals from the two arrays.

[0094] Quantitative amplification methods are also known in the art and may be used according to the methods provided herein. Methods of measurement of copy number using quantitative PCR analysis are known in the art. The known nucleotide sequence for genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR can also be used. In fluorogenic quantitative PCR, quantitation is based on the amount of fluorescence signals, e.g., TaqMan and Sybr green. Other amplification methods suitable for use according to the methods provided herein include, e.g., ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, dot PCR, and linker adapter PCR.

[0095] In some embodiments, CN or CN ratio of a gene encoding a cancer-expressed antigen is detected using hybrid capture-based sequencing (hybrid capture-based NGS), e.g., using adaptor ligation-based libraries. See, e.g., Frampton, G.M. et al. (2013) Nat. Biotech. 31:1023-1031, which is hereby incorporated by reference. In some embodiments, CN or CN ratio of a gene encoding a cancer- expressed antigen is detected using next-generation sequencing (NGS). Next-generation sequencing22MOFO-359650473Attorney Docket No: 197102019240 includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 105molecules may be sequenced simultaneously). Next generation sequencing methods suitable for use according to the methods provided herein are known in the art and include, without limitation, massively parallel short-read sequencing, template-based sequencing, pyrosequencing, real-time sequencing comprising imaging the continuous incorporation of dyelabeling nucleotides during DNA synthesis, nanopore sequencing, sequencing by hybridization, nanotransistor array based sequencing, polony sequencing, scanning tunneling microscopy (STM)-based sequencing, or nanowire -molecule sensor based sequencing. See, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31 -46, which is hereby incorporated by reference. Exemplary NGS methods and platforms include, without limitation, the HeliScope Gene Sequencing system from Helicos™ BioSciences (Cambridge, MA., USA), the PacBio® RS system from Pacific Biosciences (Menlo Park, CA, USA), massively parallel short-read sequencing such as the Solexa sequencer and other methods and platforms from Illumina™ Inc. (San Diego, CA, USA), 454 sequencing from 454 LifeSciences (Branford, CT, USA), Ion Torrent™ sequencing from ThermoFisher (Waltham, MA, USA), or the SOLiD® sequencer from Applied Biosystems (Foster City, CA, USA). Additional exemplary methods and platforms include, without limitation, the Genome Sequencer (GS) FEX™ System from Roche (Basel, CHE), the G.007 polonator system, the Solexa Genome Analyzer, HiSeq® 2500, HiSeq3000, HiSeq 4000, and NovaSeq™ 6000 platforms from Illumina™ Inc. (San Diego, CA, USA).

[0096] In some embodiments, ploidy of a sample refers to an average ploidy of two or more, five or more, 10 or more, 50 or more, or 100 or more segments of the genome. Techniques for assaying sample ploidy are known in the art. In some embodiments, ploidy is detected by sequencing, e.g., massively parallel sequencing (MPS), whole genome sequencing (WGS), or next-generation sequencing (NGS). In some embodiments, ploidy is determined by real-time polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), or droplet digital PCR (ddPCR). In some embodiments, ploidy is determined by in situ hybridization (ISH), comparative genomic hybridization (CGH), or microarray analysis.

[0097] In some embodiments, the antigen (e.g., encoded by the gene for which CN or CN ratio is calculated) is expressed by the cancer, e.g., by cells of the cancer. In some embodiments, the antigen e.g., encoded by the gene for which CN or CN ratio is calculated) is expressed on the surface of cells of the cancer. In some embodiments, the antigen (e.g., encoded by the gene for which CN or CN ratio is calculated) is bound (e.g., specifically bound) by the antibody component of an ADC of the present disclosure.

[0098] In some embodiments, the antigen (e.g., of which CN or CN ratio is calculated) is HER2. As is known in the art, HER2 and gene product(s) thereof refer to members of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. In some embodiments, HER2 refers to a23MOFO-359650473Attorney Docket No: 197102019240 human HER2 gene or gene product thereof. HER2 is also known as ERBB2, NEU, NGL, HER-2, TKR1, CD340, VSCN2, MLN19, MLN-19, C-ERB2, c-ERB-2, HER-2 / neu, and pl85(erbB2). An exemplary and non-limiting human HER2 gene is represented by NCBI Gene ID 2064. An exemplary and non-limiting human HER2 polypeptide is represented by NP_001005862.1.

[0099] In some embodiments, the antigen (e.g., of which CN or CN ratio is calculated) is c-MET. As is known in the art, c-MET and gene product(s) thereof refer to members of the receptor tyrosine kinase family. In some embodiments, c-MET refers to a human c-MET gene or gene product thereof. c-MET is also known as MET, DAI 1, HGFR, AUTS9, RCCP2, and DFNB97. An exemplary and non-limiting human c-MET gene is represented by NCBI Gene ID 4233. An exemplary and nonlimiting human c-MET polypeptide is represented by NP_000236.2.

[0100] In some embodiments, the antigen (e.g., of which CN or CN ratio is calculated) is Trop-2. As is known in the art, Trop-2 and gene product(s) thereof refer to a cell surface receptor transducing calcium signals that is expressed in certain cancers, e.g., carcinomas. In some embodiments, Trop-2 refers to a human Trop-2 gene or gene product thereof. Trop-2 is also known as tumor associated calcium signal transducer 2 (TACSTD2), EGP1, GP50, M1S1, TROP2, GA7331, and GA733-1. An exemplary and non-limiting human Trop-2 gene is represented by NCBI Gene ID 4070. An exemplary and non-limiting human Trop-2 polypeptide is represented by NP_002344.2.

[0101] In some embodiments, the antigen (e.g., of which CN or CN ratio is calculated) is FRa. As is known in the art, FRa and gene product(s) thereof refer to a member of the folate receptor family. In some embodiments, FRa refers to a human FRa gene or gene product thereof. FRa is also known as FOLR1, FBP, FOLR, FOLR1, NCFTD, and FRalpha. An exemplary and non-limiting human FRa gene is represented by NCBI Gene ID 2348. An exemplary and non-limiting human FRa polypeptide is represented by NP_000793.1.

[0102] In some embodiments, the antigen e.g., of which CN or CN ratio is calculated) is claudin 18. As is known in the art, claudin 18 and gene product(s) thereof refer to members of the claudin family of integral membrane proteins. In some embodiments, claudin 18 refers to a human claudin 18 gene or gene product thereof. Claudin 18 is also known as CLDN18, SFTA5, and SFTPJ. An exemplary and non-limiting human claudin 18 gene is represented by NCBI Gene ID 51208. An exemplary and non-limiting human claudin 18 polypeptide is represented by NP_001002026.1.

[0103] A variety of CN ratio thresholds may be used or selected by the skilled person guided by the present disclosure. In some embodiments, a CN ratio of greater than or equal to 0.5, greater than 0.5, greater than or equal to 1, greater than 1, greater than or equal to 2.5, greater than 2.5, greater than or equal to 5, greater than 5, greater than or equal to 10, greater than 10, greater than or equal to 12, greater than 12, greater than or equal to 14, greater than 14, greater than or equal to 15, greater than 15, greater than or equal to 16, greater than 16, greater than or equal to 18, greater than 18, greater than or equal to 20, or greater than 20 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising an ADC of the24MOFO-359650473Attorney Docket No: 197102019240 present disclosure (e.g., whose antibody component specifically binds the antigen). In some embodiments, the CN ratio is less than or equal to 5, less than or equal to 6, less than or equal to 7, less than or equal to 8, less than or equal to 9, or less than or equal to 10. In some embodiments, a CN ratio of a gene encoding an antigen that predicts improved outcome to treatment with an ADC targeting the antigen (e.g., whose antibody component specifically binds the antigen) is any CN ratio having an upper limit of less than or equal to 2.5, 3, 4, 5, 6, 7.5, 8, 9, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 35, or 40 and an independently selected lower limit of greater than or equal to 0.5, 1, 2.5, 3, 4, 5, 6, 7.5, 8, 9, or 10, wherein the upper limit is greater than the lower limit.

[0104] In some embodiments, a CN ratio of greater than or equal to 1, greater than 1, greater than or equal to 2.5, greater than 2.5, greater than or equal to 5, greater than 5, greater than or equal to 10, greater than 10, greater than or equal to 12, greater than 12, greater than or equal to 14, greater than 14, greater than or equal to 15, greater than 15, greater than or equal to 16, greater than 16, greater than or equal to 18, greater than 18, greater than or equal to 20, or greater than 20 (and optionally less than 5, 6, 7, 8, 9, or 10) of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising an ADC of the present disclosure, e.g., as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC. In some embodiments, improved outcome comprises longer progression-free survival (PFS) e.g., rwPFS), longer overall survival (OS) (e.g., rwOS), and / or longer time to treatment discontinuation (TTD).

[0105] In some embodiments, a CN ratio of less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5 (and optionally greater than 0) of the gene encoding the antigen in the sample predicts no improved outcome associated with treating the individual with a treatment comprising an ADC of the present disclosure, e.g., as compared to outcome of treating an individual with a therapeutic agent other than an ADC. In some embodiments, a CN of less than 2, less than 1.8, less than 1.6, less than 1.5, less than 1.2, or less than 1 (and optionally greater than 0) of the antigen in the sample predicts no improved outcome associated with treating the individual with a treatment comprising an ADC of the present disclosure, e.g., as compared to outcome of treating an individual with a therapeutic agent other than an ADC. In some embodiments, the therapeutic agent is a standard of care treatment or chemotherapeutic agent that does not comprise an ADC.

[0106] In some embodiments, the methods comprise performing DNA sequencing on a sample obtained from an individual (e.g., an individual having cancer or being treated for cancer) to determine CN ratio of a gene encoding an antigen. In some embodiments, the methods further comprise identifying a candidate treatment for a cancer in an individual, based at least in part on the CN ratio. In some embodiments, the candidate treatment comprises an ADC (e.g., when the CN ratio is greater than or equal to a CN ratio threshold value). In some embodiments, the candidate treatment comprises a therapeutic agent other than an ADC (e.g., when the CN ratio is less than a CN ratio threshold value).25MOFO-359650473Attorney Docket No: 197102019240

[0107] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based at least in part on CN ratio of a gene encoding an antigen in the sample, wherein the one or more treatment options comprise an ADC, e.g., targeting the antigen.

[0108] In some embodiments of any of the methods provided herein, responsive to acquisition of knowledge or detection of CN ratio of a gene encoding an antigen in a sample from an individual (e.g., an individual having cancer or being treated for cancer): (i) the individual is classified as a candidate to receive a treatment comprising an ADC, e.g., targeting the antigen; (ii) the individual is identified as likely to respond or have favorable outcome in response to a treatment that comprises an ADC, e.g., targeting the antigen; (iii) the individual is identified as unlikely to respond to a treatment that comprises an ADC, e.g., targeting the antigen; and / or (iv) the individual is identified as likely to have a poor prognosis or outcome in response to a treatment that comprises an ADC, e.g., targeting the antigen.

[0109] In some embodiments, responsive to acquisition of knowledge or detection of CN ratio of a gene encoding an antigen in a sample from an individual (e.g., an individual having cancer or being treated for cancer), the methods comprise administering to the individual an effective amount of a treatment that comprises an ADC, e.g., targeting the antigen. In some embodiments, responsive to acquisition of knowledge or detection of CN ratio of a gene encoding an antigen in a sample from an individual (e.g., an individual having cancer or being treated for cancer), the methods comprise administering to the individual an effective amount of a therapeutic agent other than an ADC (e.g., a standard of care treatment or chemotherapeutic agent that does not comprise an ADC).

[0110] In some embodiments of any of the methods provided herein, the methods further comprise generating a report comprising one or more treatment options identified for the individual based, at least in part, on CN ratio of a gene encoding an antigen in a sample from the individual, wherein the one or more treatment options comprise an ADC, e.g., targeting the antigen. In some embodiments, the methods further comprise generating a report comprising one or more treatment options identified for the individual based, at least in part, on CN ratio of a gene encoding an antigen in a sample from the individual, wherein the one or more treatment options comprise a therapeutic agent other than an ADC (e.g., a standard of care treatment or chemotherapeutic agent that does not comprise an ADC).

[0111] In some embodiments, acquiring knowledge of CN ratio of a gene encoding an antigen in a sample comprises detecting CN or CN ratio of a gene encoding the antigen in the sample. In some embodiments, acquiring knowledge of CN ratio of a gene encoding an antigen in a sample comprises acquiring knowledge of or detecting ploidy of the sample.

[0112] In some embodiments, the methods of the disclosure further comprise providing an assessment of the CN or CN ratio of a gene encoding an antigen, e.g., in a sample.26MOFO-359650473Attorney Docket No: 197102019240Antibody-drug conjugates (ADCs)

[0113] In some embodiments of any of the methods provided herein, the anti-cancer therapy is an ADC.

[0114] In some embodiments, the treatment comprising the ADC is an adjuvant or neoadjuvant treatment. In some embodiments, a treatment comprising an ADC is a first-line (IL) treatment. In some embodiments, a treatment comprising an ADC is a second-line or higher (2L+) treatment. In some embodiments, a treatment comprising an ADC is a third-line or higher (3L+) treatment. In some embodiments, a treatment comprising an ADC is a second-line or third-line treatment.

[0115] In some embodiments, the ADC exhibits a “bystander effect” or “bystander killing.” Some ADCs have been shown to exhibit a bystander effect, e.g., ADC-mediated cell killing of cancer cells that express minimal or no antigen targeted by the ADC but are nonetheless killed by the ADC, e.g., by escape of the payload from cells that do express the antigen (e.g., proximal to the “bystander” cells), escape of the payload from the extracellular space, and / or changes to the tumor microenvironment mediated by the payload / ADC. See, e.g., Staudacher, A.H. and Brown, M.P.(2017) Br J Cancer. 117(12): 1736-1742. In some embodiments, the ADC has a membrane-permeable pay load or a transmembrane pay load. In some embodiments, the ADC has a cleavable linker.

[0116] A variety of ADCs whose antibody components target different cancer antigens are known in the art and approved for clinical use or undergoing clinical testing.

[0117] In some embodiments, the antibody component of an ADC of the present disclosure binds (e.g., specifically binds) a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, the ADC is trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), disitamab vedotin (RC48), trastuzumab duocarmazine (SYD985), XMT-1522, ZRC-3256, MRG002, ARX788, B DC-1001, A166, FS-1502, SHR-A1201, DP303c, BI-CON-02, ALT-P7, DX126-262, ZW49, HS630, B003, SBT6050, SHR-A1811, MT-5111, GQ1001, PF-06804103, or T-PNU.

[0118] In some embodiments, a cancer to be treated or analyzed by the methods of the present disclosure is HER2(+). In some embodiments, the cancer expresses HER2 or shows HER2 expression at a level of 3 or higher, as measured by immunohistochemistry (IHC). In some embodiments, the cancer is positive for HER2 expression by in situ hybridization (ISH) and shows HER2 expression at a level of 2 or higher as measured by IHC.

[0119] In some embodiments, a cancer to be treated or analyzed by the methods of the present disclosure is HER2-low or HER2(-). In some embodiments, the cancer expresses HER2 or shows HER2 expression at a level of 0 as measured by IHC. In some embodiments, the cancer is negative for HER2 expression by ISH and shows HER2 expression at a level of 1 or higher or 2 or higher as measured by IHC.

[0120] Certain assays are commonly used in the art to measure expression of a cancer cell surface antigen such as HER2. Commonly known assays include immunohistochemistry (IHC) and in situ27MOFO-359650473Attorney Docket No: 197102019240 hybridization (ISH). For example, standard guidelines for assessing HER2 by IHC and ISH are known in the art; see, e.g., Wolff, A.C. et al. (2018) Arch Pathol Lab Med. 142(11): 1364-1382.

[0121] In some embodiments, an individual to be treated by the methods of the present disclosure has received prior treatment with a HER2-targeted therapy. A variety of HER2-targeted therapies are known in the art. In other embodiments, the individual has not received prior treatment with a HER2- targeted therapy. In some embodiments, the HER2-targeted therapy comprises trastuzumab and / or pertuzumab.

[0122] In some embodiments, the antibody component of an ADC of the present disclosure binds (e.g., specifically binds) a c-mesenchymal-epithelial transition (c-MET) polypeptide e.g., a human c- MET polypeptide). ADCs targeting c-MET are known in the art; see, e.g., Han, Y. et al. (2024) JTO Clin Res Rep. 5(2): 100630. Non-limiting examples of ADCs targeting c-MET include, without limitation, telisotuzumab vedotin (Teliso-V, ABBV-399), RC108, and HRA00129-C004.

[0123] In some embodiments, the antibody component of an ADC of the present disclosure binds (e.g., specifically binds) a trophoblast cell surface antigen 2 (Trop-2) polypeptide (e.g., a human Trop- 2 polypeptide). ADCs targeting Trop-2 are known in the art; see, e.g., Shastry, M. et al. (2022) Breast 66:169-177. Non-limiting examples of ADCs targeting Trop-2 include, without limitation, sacituzumab govitecan, datopotamab deruxtecan (Dato-DXd), SKB-264, BIO-106, DB-1305, ESG- 401, BAT-8008, BL-M02D1, DAC-002 (JS108), FDA018, and SHR-A1921.

[0124] In some embodiments, the antibody component of an ADC of the present disclosure binds (e.g., specifically binds) a folate receptor alpha (FRa) polypeptide (e.g., a human FRa polypeptide). Non-limiting examples of ADCs targeting FRa include, without limitation, mirvetuximab soravtansine, luveltamab tazevibulin (STRO-002), and MORAb-202.

[0125] In some embodiments, the antibody component of an ADC of the present disclosure binds (e.g., specifically binds) a claudin 18 polypeptide (e.g., a human claudin 18 polypeptide). Nonlimiting examples of ADCs targeting claudin 18 include, without limitation, CMG901, IBI343, SKB315 (MK-1200), TORL-2-307-ADC, SYSA1801, AZD0901 (CMG901), ATG-022, SOT102, and EO-3021.

[0126] In some embodiments, the methods of the present disclosure comprise administering to the individual a therapeutic agent other than an ADC, e.g., if the CN ratio of a gene encoding an antigen is less than a threshold CN ratio of the present disclosure. In some embodiments, the therapeutic agent other than an ADC is a standard of care treatment or a chemotherapeutic agent. For example, the therapeutic agent could include an aromatase inhibitor, immune checkpoint inhibitor, selective estrogen receptor degrader (SERD), poly(ADP-ribose) polymerase inhibitor (PARPi), or cyclin- dependent kinase inhibitor (e.g., CD A / 6 inhibitor). Non-limiting examples of CDK inhibitors include palbociclib, ribociclib, and abemaciclib, as well as pharmaceutically acceptable salts thereof. Non-limiting examples of PARPis include talazoparib, rucaparib, niraparib, and olaparib, as well as pharmaceutically acceptable salts thereof. Non-limiting examples of aromatase inhibitors include28MOFO-359650473Attorney Docket No: 197102019240 aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxy androstenedione, 1, 4, 6-androstatrien-3, 17-dione (ATD), 4- Androstene-3, 6, 17-trione (“6- OXO”), or a pharmaceutically acceptable salt thereof. Non-limiting examples of SERDs include fulvestrant, elacestrant, amcenestrant, camizestrant, giredestrant, rintodestrant, imlunestrant, ZB -716, Zn-c5, LSZ102, LY3484356, or D-0502, or a pharmaceutically acceptable salt thereof. Such nonADC therapeutic agents may be selected, e.g., based on other characteristics of the cancer to be treated. For example, an aromatase inhibitor or SERD could be used to treat hormone receptor positive (HR+) breast cancer, or a PARPi could be used to treat breast cancer with a BRCA1 / BRCA2 or PALB2 mutation.

[0127] In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the ADC, further comprise an additional anti-cancer therapy. In some embodiments of any of the methods provided herein, the treatment or the one or more treatment options, e.g., the ADC, further comprise administering an additional anti-cancer therapy to the individual, e.g., administering an ADC in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is any anti-cancer therapy known in the art or described herein. In some embodiments, the additional anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the additional anti-cancer therapy is an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy is a PD-1-, a CTLA4-, or a PD-L1 -targeted agent. In some embodiments, the additional anti-cancer therapy is a heat shock protein 90 inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018; Pall, Current opinion in oncology vol. 27,2 (2015): 118-24), an EGFR inhibitor (Golding et al., Molecular cancer vol. 17,1 52, 2018), a SHP2 inhibitor (Dardaei et al., Nature medicine vol. 24,4 (2018): 512-517), a MEK inhibitor (Shrestha et al., Scientific reports vol. 9,1 18842, 2019; Shrestha et al., The Journal of pharmacology and experimental therapeutics vol. 374,1 (2020): 134-140), an IGF-1R inhibitor (George, Journal of hematology & oncology vol. 12,1 80, 2019), a vascular endothelial growth factor (VEGF)-targeted therapy (Makimoto et al., Acta medica Okayama vol. 74,5 (2020): 371-379; Gristina et al., Pharmaceuticals (Basel, Switzerland) vol. 13,12 474, 2020), an mTOR inhibitor (Kim et al., Anticancer research vol. 40,3 (2020): 1395-1403), or any combination thereof.

[0128] As is known in the art, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include, e.g., CTLA4, PD-L1, PD-1, PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CEACAM,29MOFO-359650473Attorney Docket No: 197102019240LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-CD, CD273), CTLA-4 (CD152), HVEM, BTLA (CD272), a killer-cell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM-1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, A2AR, GITR (CD357), CD80 (B7-1), CD86 (B7-2), CD276 (B7-H3), VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, 0X40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d, OX40L (CD134L), PVR (NECL5, CD155), SIRPa, MICA / B, and / or arginase. In some embodiments, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anticancer immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD-1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitory molecule (e.g., a CTLA4 antagonist (e.g., an anti-CTLA4 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see, e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012). In some embodiments, known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0129] In some embodiments, the checkpoint inhibitor is a PD-L1 axis binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1,” "PDCD1 LG1," "CD274," "B7-H," and "PDL1." An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1 LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.30MOFO-359650473Attorney Docket No: 197102019240

[0130] In some embodiments, the checkpoint inhibitor is a PD-1 binding antagonist / inhibitor. In some embodiments, the PD-1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific embodiment, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In some embodiments, the checkpoint inhibitor is a PD-L1 binding antagonist / inhibitor. In some embodiments, a PD-L1 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L1 to its binding ligands. In a specific embodiment, PD-L1 binding partners are PD-1 and / or B7-1. In some embodiments, the checkpoint inhibitor is a PD-L2 binding antagonist / inhibitor. In some embodiments, the PD-L2 binding antagonist / inhibitor is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific embodiment, the PD-L2 binding ligand partner is PD-1. The antagonist or inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1, PD-L1, or PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0131] In some instances, the PD-1 binding antagonist or inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, as described below. In some instances, the anti-PD-1 antibody is one or more of MDX-1 106 (nivolumab), MK-3475 (pembrolizumab, e.g., Keytruda®), MEDI-0680 (AMP-514), PDR001, REGN2810, MGA-012, JNJ- 63723283, BI 754091, BGB-108, BGB-A317, JS-001, STI-Al l 10, INCSHR-1210, PF-06801591, TSR-042, AM0001, ENUM 244C8, ENUM 388D4, cemiplimab, or dostarlimab. In other instances, the PD-1 binding antagonist or inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some instances, the PD-1 binding antagonist or inhibitor is AMP-224. Other examples of anti-PD-1 antibodies include, but are not limited to, MEDI-0680 (AMP-514; AstraZeneca), PDR001 (CAS Registry No. 1859072-53-9; Novartis), REGN2810 (e.g., LIBTAYO® or cemiplimab-rwlc; Regeneron), BGB-108 (BeiGene), BGB-A317 (BeiGene), BI 754091, JS-001 (Shanghai Junshi), STI-Al l 10 (Sorrento), INCSHR-1210 (Incyte), PF-06801591 (Pfizer), TSR-042 (also known as ANB011; Tesaro / AnaptysBio), AM0001 (ARMO Biosciences), ENUM 244C8 (Enumeral Biomedical Holdings), or ENUM 388D4 (Enumeral Biomedical Holdings). In some embodiments, the PD-1 axis binding antagonist or inhibitor comprises tislelizumab (BGB- A317), BGB-108, STI-Al l 10, AM0001, BI 754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP- 514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO-4538), spartalizumab (PDR001), pembrolizumab (MK-3475, SCH 900475, e.g., Keytruda®), PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostarlimab (TSR-042, ANB011), FITC-YT-16 (PD-1 binding peptide), APL-501 or CBT-501 or genolimzumab (GB-226), AB-122, AK105, AMG 404, BCD-100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP-224 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (ABBV-181), AK-103, BAT-1306,31MOFO-359650473Attorney Docket No: 197102019240CS-1003, AM-0001, TILT-123, BH-2922, BH-2941, BH-2950, ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI 11-003, IKT-202, MCLA-134, MT-17000, PEGMP-7, PRS-332, RXI-762, STI- 1110, VXM-10, XmAb-23104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7769, CAB PD-1 Abs, AK-123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD- 288, SG-001, BY-24.3, CB-201, IBI-319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, or CCX- 4503, or derivatives thereof, or an antibody that competes with any of the preceding.

[0132] In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-1. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule that inhibits PD-L1 and VISTA or PD-L1 and TIM3. In some embodiments, the PD-L1 binding antagonist or inhibitor is CA-170 (also known as AUPM-170). In some embodiments, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD- L1 antibody can bind to a human PD-L1, for example a human PD-L1 as described above herein and / or as shown in UniProtKB / Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof. In some embodiments, the PD-L1 binding antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin.

[0133] In some instances, the PD-L1 binding antagonist or inhibitor is an anti-PD-Ll antibody, for example, as described below. In some instances, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-L1 and PD-1, and / or between PD-L1 and B7-1. In some instances, the anti-PD- Ll antibody is a monoclonal antibody. In some instances, the anti-PD-Ll antibody is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some instances, the anti-PD- E1 antibody is a humanized antibody. In some instances, the anti-PD-El antibody is a human antibody. In some instances, the anti-PD-El antibody is selected from YW243.55.S70, MPDE3280A (atezolizumab), MDX-1 105, MEDI4736 (durvalumab), MSB0010718C (avelumab), EY3300054, STI-A1014, KN035, FAZ053, or CX-072. In some embodiments, the PD-E1 axis binding antagonist or inhibitor comprises atezolizumab, avelumab, durvalumab (imfinzi), BGB-A333, SHR-1316 (HTI- 1088), CK-301, BMS-936559, envafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS-936559), EY3300054, STI-A1014, FAZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088 (HTI-131, SHR-1316), MSB-2311, AK- 106, AVA-004, BBI-801, CA-327, CBA-0710, CBT-502, FPT-155, IKT-201, IKT-703, 10-103, JS-003, KD-033, KY-1003, MCEA-145, MT-5050, SNA-02, BCD-135, APE-502 (CBT-402 or TQB2450), IMC-001, KD-045, INBRX-105, KN-046, IMC-2102, IMC-2101, KD-005, IMM-2502, 89Zr-CX-072, 89Zr-DFO-6El l, KY-1055, MEDI-1109, MT-5594, SL-279252, DSP-106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, or FS-118, or a derivative thereof, or an antibody that competes with any of the preceding.

[0134] In some embodiments, the checkpoint inhibitor is an antagonist or inhibitor of CTEA4. In some embodiments, the checkpoint inhibitor is a small molecule antagonist or inhibitor of CTEA4. In some embodiments, the checkpoint inhibitor is an anti-CTEA4 antibody. CTEA4 is part of the CD28-32MOFO-359650473Attorney Docket No: 197102019240B7 immunoglobulin superfamily of immune checkpoint molecules that acts to negatively regulate T cell activation, particularly CD28-dependent T cell responses. CTLA4 competes for binding to common ligands with CD28, such as CD80 (B7-1) and CD86 (B7-2), and binds to these ligands with higher affinity than CD28. Blocking CTLA4 activity (e.g., using an anti-CTLA4 antibody) is thought to enhance CD28-mediated costimulation (leading to increased T cell activation / priming), affect T cell development, and / or deplete Tregs (such as intratumoral Tregs). In some embodiments, the CTLA4 antagonist or inhibitor is a small molecule, a nucleic acid, a polypeptide (e.g., antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the CTLA-4 antagonist or inhibitor comprises ipilimumab (IBI310, BMS-734016, MDX010, MDX-CTLA4, MEDI4736), tremelimumab (CP-675, CP-675,206), APL-509, AGEN1884, CS1002, AGEN1181, Abatacept (Orencia, BMS- 188667, RG2077), BCD-145, ONC-392, ADU-1604, REGN4659, ADG116, KN044, KN046, or a derivative thereof, or an antibody that competes with any of the preceding.

[0135] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 antagonist or inhibitor (e.g., an antibody, an antibody conjugate, or an antigen-binding fragment thereof). In some embodiments, the LAG-3 antagonist or inhibitor comprises a small molecule, a nucleic acid, a polypeptide (e.g., an antibody), a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the LAG-3 antagonist or inhibitor comprises a small molecule. In some embodiments, the LAG-3 antagonist or inhibitor comprises a LAG-3 binding agent. In some embodiments, the LAG-3 antagonist or inhibitor comprises an antibody, an antibody conjugate, or an antigen-binding fragment thereof. In some embodiments, the LAG-3 antagonist or inhibitor comprises eftilagimod alpha (IMP321, IMP-321, EDDP-202, EOC-202), relatlimab (BMS-986016), GSK2831781 (IMP-731), LAG525 (IMP701), TSR-033, EVIP321 (soluble LAG-3 protein), BI 754111, IMP761, REGN3767, MK-4280, MGD-013, XmAb22841, INCAGN-2385, ENUM-006, AVA-017, AM-0003, iOnctura anti-LAG-3 antibody, Arcus Biosciences LAG-3 antibody, Sym022, a derivative thereof, or an antibody that competes with any of the preceding.

[0136] In some embodiments, the immune checkpoint inhibitor is monovalent and / or monospecific. In some embodiments, the immune checkpoint inhibitor is multivalent and / or multispecific.

[0137] In some embodiments of any of the methods provided herein, the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is biliary tract cancer, breast cancer, gastric cancer, colorectal cancer, or lung cancer. In some embodiments, the cancer is advanced or metastatic.Samples

[0138] In some embodiments of any of the methods provided herein, the sample is a sample described below. In some embodiments, the sample is obtained from the individual or from the33MOFO-359650473Attorney Docket No: 197102019240 cancer. In some embodiments, the methods further comprise obtaining the sample, e.g., from the individual or from the cancer. In some embodiments, the sample comprises a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some embodiments, the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof. In some embodiments, the fusion nucleic acid molecule or polypeptide is detected in a tissue biopsy sample, in a liquid biopsy sample, or in both a tissue biopsy sample and a liquid biopsy sample, from the individual.

[0139] In some embodiments, CN, ploidy, and / or CN ratio of a gene is / are detected in a cancer or tumor cell, e.g., using tumor tissue, such as from a tumor biopsy or other tumor specimen; in a circulating cancer or tumor cell, e.g., using a liquid biopsy, such as from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva; or in circulating tumor DNA (ctDNA), e.g., using a liquid biopsy, such as from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.

[0140] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (i) obtaining a sample from an individual (e.g., an individual having, suspected of having, or determined to have cancer), (ii) extracting nucleic acid molecules e.g., a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid molecules) from the sample, (iii) ligating one or more adapters to the nucleic acid molecules extracted from the sample (e.g., one or more amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences), (iv) amplifying the nucleic acid molecules (e.g., using a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique), (v) capturing nucleic acid molecules from the amplified nucleic acid molecules (e.g., by hybridization to one or more bait molecules, where the bait molecules each comprise one or more nucleic acid molecules (e.g., capture nucleic acid molecules) that each comprise a region that is complementary to a region of a captured nucleic acid molecule), (vi) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, and (vii) generating, displaying, transmitting, and / or delivering a report (e.g. , an electronic, web-based, or paper report) to the individual (or patient), a caregiver, a healthcare provider, a physician, an34MOFO-359650473Attorney Docket No: 197102019240 oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[0141] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a plurality of nucleic acid molecules obtained from a sample from an individual e.g., an individual having cancer), wherein the plurality of nucleic acid molecules comprises nucleic acid molecules corresponding to a gene encoding a cancer-expressed antigen of the disclosure; (b) ligating one or more adapters onto one or more nucleic acid molecules from the plurality of nucleic acid molecules; (c) amplifying the one or more ligated nucleic acid molecules from the plurality of nucleic acid molecules; (d) capturing amplified nucleic acid molecules from the amplified nucleic acid molecules; (e) sequencing, e.g., by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the cancer-expressed antigen nucleic acid molecule; (f) analyzing the plurality of sequence reads; and (g) based on the analysis, detecting CN or CN ratio of the gene encoding the cancer-expressed antigen nucleic acid molecule in the sample. In some embodiments, the methods further comprise receiving, e.g., at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, the analyzing the plurality of sequence reads comprises identifying, e.g., using the one or more processors, the presence or absence of sequence reads corresponding to the fusion nucleic acid molecule. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.

[0142] In some embodiments of any of the methods provided herein, the methods may comprise one or more of the steps of: (a) providing a sample from an individual (e.g., an individual having, suspected of having or determined to have cancer), wherein the sample comprises a plurality of nucleic acid molecules; (b) preparing a nucleic acid sequencing library from the plurality of nucleic acid molecules in the sample; (c) amplifying said library; (d) selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to a gene encoding a cancer- expressed antigen of the disclosure in said library to produce an enriched sample; (e) sequencing the enriched sample, thereby producing a plurality of sequence reads; (f) analyzing the plurality of sequence reads for the presence of the cancer-expressed antigen nucleic acid molecule; (g) detecting, based on the analyzing step, CN or CN ratio of the gene encoding the cancer-expressed antigen nucleic acid molecule in the sample from the individual.

[0143] In some embodiments of any of the methods provided herein, the plurality of nucleic acid molecules comprises a mixture of cancer nucleic acid molecules and non-cancer nucleic acid molecules. In some embodiments, the cancer nucleic acid molecules are derived from a tumor portion35MOFO-359650473Attorney Docket No: 197102019240 of a heterogeneous tissue biopsy sample, and the non-cancer nucleic acid molecules are derived from a normal portion of the heterogeneous tissue biopsy sample. In some embodiments, the sample comprises a liquid biopsy sample, and the cancer nucleic acid molecules are derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample; and the non-cancer nucleic acid molecules are derived from a non-tumor fraction of the liquid biopsy sample or a cell-free DNA (cfDNA) fraction of the liquid biopsy sample.

[0144] In some embodiments of any of the methods, the one or more adapters comprise amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the one or more adapters comprise one or more sample index sequences. As is known in the art, sample indexes allow the sequencing of multiple samples on the same instrument flow cell or chip (i.e., multiplexing). Sample indexes are typically between about 8 and about 10 bases in length, and comprise a nucleotide sequence specific to a sample that is used to assign sequence reads to the correct sample during data analysis. In some embodiments, the one or more adapters comprise one or more unique molecule identifiers (UMIs). As is known in the art, UMIs comprise short nucleotide sequences that include a unique barcode that is incorporated into each molecule in a given sample library. UMIs are useful for identifying PCR duplicates created during library amplification steps, and / or for reducing the rate of false-positive variant calls and increasing variant detection, since variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.

[0145] In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to a gene encoding a cancer-expressed antigen and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the captured nucleic acid molecules are captured from the amplified nucleic acid molecules by hybridization to one or more bait molecules. In some embodiments, the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the sequencer comprises a next generation sequencer.

[0146] In some embodiments of any of the methods provided herein, the methods further comprise selectively enriching for one or more nucleic acids in the sample comprising nucleotide sequences corresponding to a gene encoding a cancer-expressed antigen nucleic acid molecule of the disclosure.36MOFO-359650473Attorney Docket No: 197102019240In some embodiments, the selectively enriching produces an enriched sample. In some embodiments, the selectively enriching comprises: (a) combining one or more bait molecules with the sample, thereby hybridizing the one or more bait molecules to one or more nucleic acids in the sample comprising nucleotide sequences corresponding to the gene encoding the cancer-expressed antigen nucleic acid molecule and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids to produce the enriched sample. In some embodiments, the selectively enriching comprises amplifying the one or more nucleic acids comprising nucleotide sequences corresponding to the gene encoding the cancer-expressed antigen nucleic acid molecule using a polymerase chain reaction (PCR) to produce an enriched sample. In some embodiments, the methods further comprise sequencing the enriched sample.

[0147] In some embodiments of any of the methods provided herein, the methods further comprise generating a molecular profile for the individual or the sample, based, at least in part, on detecting the CN or CN ratio of the gene encoding the cancer-expressed antigen nucleic acid molecule. In some embodiments, the molecular profile for the individual or sample further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile further comprises results from a nucleic acid sequencing-based test.

[0148] In some embodiments of any of the methods provided herein, the methods further comprise selecting a treatment, administering a treatment, or applying a treatment to the individual based on the generated molecular profile, wherein the treatment comprises an ADC, e.g., as described herein.

[0149] In some embodiments of any of the methods provided herein, the methods further comprise generating a report indicating the CN or CN ratio of the gene encoding the cancer-expressed antigen in the sample. In some embodiments, the methods further comprise generating, by the one or more processors, a report indicating the CN or CN ratio of the gene encoding the cancer-expressed antigen in the sample. In some embodiments, the methods further comprise transmitting the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network or a peer- to-peer connection.

[0150] The disclosed methods may be used with any of a variety of samples, e.g., as described in further detail below. For example, in some instances, the sample may comprise a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some instances, the sample may be a liquid biopsy sample and may comprise blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell- free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0151] In some instances, the nucleic acid molecules extracted from a sample may comprise a mixture of tumor or cancer nucleic acid molecules and non-tumor or non-cancer nucleic acid37MOFO-359650473Attorney Docket No: 197102019240 molecules. In some instances, the tumor nucleic acid molecules may be derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-tumor nucleic acid molecules may be derived from a normal portion of the heterogeneous tissue biopsy sample. In some instances, the sample may comprise a liquid biopsy sample, and the tumor or cancer nucleic acid molecules may be derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample while the non-tumor or noncancer nucleic acid molecules may be derived from a non-tumor or non-cancer, cell-free DNA (cfDNA) fraction of the liquid biopsy sample. In some embodiments of any of the methods provided herein, the method further comprises determining the circulating tumor DNA (ctDNA) fraction of a liquid biopsy sample.

[0152] A variety of materials can be the source of, or serve as, samples for use in any of the methods of the disclosure. For example, the sample can be, or be derived from: solid tissue such as from a fresh, frozen and / or preserved organ, tissue sample, biopsy (e.g., tumor, tissue or liquid biopsy), resection, smear, or aspirate; scrapings; bone marrow or bone marrow specimens; a bone marrow aspirate; blood or any blood constituents; blood cells; bodily fluids such as cerebrospinal fluid, amniotic fluid, urine, saliva, sputum, peritoneal fluid or interstitial fluid; pleural fluid; ascites; tissue or fine needle biopsy samples; surgical specimens; cell-containing body fluids; free-floating nucleic acids; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as ductal lavages or bronchoalveolar lavages; cells from any time in gestation or development of an individual; cells from a cancer or tumor; other body fluids, secretions, and / or excretions, and / or cells therefrom. In some embodiments, a sample is or comprises cells obtained from an individual. In some embodiments, the sample is or is derived from blood or blood constituents, e.g., obtained from a liquid biopsy. In some embodiments, the sample is or is derived from a tumor sample. In some embodiments, the sample is or comprises biological tissue or fluid. In some embodiments, the sample can contain compounds that are not naturally intermixed with the source of the sample in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. In some embodiments, the sample is preserved as a frozen sample or as a formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. In some embodiments, the sample comprises circulating tumor cells (CTCs).

[0153] In one embodiment, the sample comprises one or more cells associated with a tumor, e.g., tumor cells or tumor-infiltrating lymphocytes (TIL). In one embodiment, the sample includes one or more premalignant or malignant cells. In one embodiment, the sample is acquired from a hematologic malignancy (or pre-malignancy), e.g., a hematologic malignancy (or pre-malignancy) described herein. In one embodiment, the sample is acquired from a cancer, such as a cancer described herein. In some embodiments, the sample is acquired from a solid tumor, a soft tissue tumor or a metastatic lesion. In other embodiments, the sample includes tissue or cells from a surgical margin. In one embodiment, the sample is or is acquired from a liquid biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample includes cell-free DNA (cfDNA)38MOFO-359650473Attorney Docket No: 197102019240 and / or circulating tumor DNA (ctDNA), e.g., from a biopsy of blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In another embodiment, the sample includes one or more circulating tumor cells (CTCs) (e.g., a CTC acquired from a blood sample). In one embodiment, the sample is a cell not associated with a tumor or cancer, e.g., a non-tumor or non-cancer cell or a peripheral blood lymphocyte.

[0154] In some embodiments, a sample is a primary sample obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by a method chosen from biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, or collection of body fluid (e.g., blood, lymph, or feces). In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. Such a processed sample may comprise, for example, nucleic acids (e.g., for use in any of the methods provided herein) extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification methods, reverse transcription of mRNA, or isolation and / or purification of certain components such as nucleic acids.

[0155] In some embodiments, the sample comprises nucleic acids, e.g., genomic DNA, cDNA, or mRNA. In some embodiments, the sample comprises cell-free DNA (cfDNA). In some embodiments, the sample comprises cell-free RNA (cfRNA). In some embodiments, the sample comprises circulating tumor DNA (ctDNA). In certain embodiments, the nucleic acids are purified or isolated e.g., removed from their natural state). In some embodiments, the sample comprises tumor or cancer nucleic acids, such as nucleic acids from a tumor or cancer sample, e.g., genomic DNA, RNA, or cDNA derived from RNA, or from a liquid biopsy, e.g., ctDNA from blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In certain embodiments, a tumor or cancer nucleic acid sample, or a ctDNA sample, is purified or isolated (e.g., it is removed from its natural state).

[0156] In some embodiments, the sample is obtained from an individual having a cancer, such as a cancer described herein. In some embodiments, the sample comprises nucleic acid molecule(s) corresponding to a gene encoding a cancer-expressed antigen (or fragment thereof) of the disclosure.

[0157] In some embodiments, the methods provided herein comprise generating a report, and / or providing a report to party.

[0158] In some embodiments, a report according to the present disclosure comprises information about one or more of: a cancer-expressed antigen (or a gene encoding the cancer-expressed antigen) of the disclosure; a CN or CN ratio of a gene encoding a cancer-expressed antigen of the disclosure; a cancer of the disclosure; or a treatment, a therapy, or one or more treatment options for an individual having a cancer (e.g., an ADC), such as a cancer of the disclosure.

[0159] In some embodiments, a report according to the present disclosure comprises information about the presence or absence of a cancer-expressed antigen nucleic acid molecule or polypeptide of the disclosure in a sample obtained from an individual, such as an individual having a cancer, e.g., a39MOFO-359650473Attorney Docket No: 197102019240 cancer provided herein. In one embodiment, a report according to the present disclosure indicates that a cancer-expressed antigen of the disclosure is present in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates the CN or CN ratio of a gene encoding a cancer-expressed antigen of the disclosure in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that a cancer-expressed antigen of the disclosure is not present in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that a cancer-expressed antigen of the disclosure has been detected in a sample obtained from the individual. In one embodiment, a report according to the present disclosure indicates that a cancer-expressed antigen of the disclosure has not been detected in a sample obtained from the individual. In some embodiments, the report comprises an identifier for the individual from which the sample was obtained.

[0160] In some embodiments, the report includes information on the role of a cancer-expressed antigen of the disclosure in disease, such as in cancer. Such information can include one or more of: information on prognosis of a cancer, such as a cancer provided herein; information on resistance of a cancer, such as a cancer provided herein to one or more treatments (e.g., comprising an ADC); information on potential or suggested therapeutic options (e.g., such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein); or information on therapeutic options that should be avoided (e.g., an ADC). In some embodiments, the report includes information on the likely effectiveness, acceptability, and / or advisability of applying a therapeutic option (e.g., such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein) to an individual having a cancer, such as a cancer provided herein. In some embodiments, the report includes information or a recommendation on the administration of a treatment (e.g., an ADC provided herein, or a treatment selected or identified according to the methods provided herein). In some embodiments, the information or recommendation includes the dosage of the treatment and / or a treatment regimen (e.g., in combination with other treatments, such as a second therapeutic agent). In some embodiments, the report comprises information or a recommendation for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more treatments.

[0161] Also provided herein are methods of generating a report according to the present disclosure. In some embodiments, a report according to the present disclosure is generated by a method comprising one or more of the following steps: obtaining a sample, such as a sample described herein, from an individual, e.g., an individual having a cancer, such as a cancer provided herein; detecting a CN or CN ratio of a gene encoding a cancer-expressed antigen in the sample, or acquiring knowledge of CN or CN ratio of a gene encoding a cancer-expressed antigen in the sample; and generating a report. In some embodiments, a report generated according to the methods provided herein comprises one or more of: information about the CN or CN ratio of a gene encoding a cancer-expressed antigen of the disclosure in the sample; an identifier for the individual from which the sample was obtained;40MOFO-359650473Attorney Docket No: 197102019240 information on the role of the gene or cancer-expressed antigen in disease (e.g., such as in cancer); information on prognosis, resistance, or potential or suggested therapeutic options (such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein); information on the likely effectiveness, acceptability, or the advisability of applying a therapeutic option (such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein) to the individual; a recommendation or information on the administration of a treatment (such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein); or a recommendation or information on the dosage or treatment regimen of a treatment (such as an ADC provided herein, or a treatment selected or identified according to the methods provided herein), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0162] A report according to the present disclosure may be in an electronic, web-based, or paper form. The report may be provided to an individual or a patient (e.g., an individual or a patient having, suspected of having, or being tested for a cancer, such as a cancer provided herein), or to an individual or entity other than the individual or patient, such as one or more of a caregiver, a physician, an oncologist, a hospital, a clinic, a third party payor, an insurance company, or a government entity. In some embodiments, the report is provided or delivered to the individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from detecting a CN or CN ratio of a cancer-expressed antigen in a sample obtained from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from acquiring knowledge of CN or CN ratio of a gene encoding a cancer- expressed antigen in a sample obtained from the individual.E. Software, Systems, and Devices

[0163] In some other aspects, provided herein are non-transitory computer-readable storage media. In some embodiments, the non-transitory computer-readable storage media comprise one or more programs for execution by one or more processors of a device, the one or more programs including instructions which, when executed by the one or more processors, cause the device to perform the method according to any of the embodiments described herein.

[0164] FIG. 10 illustrates an example of a computing device or system in accordance with one embodiment. Device 1000 can be a host computer connected to a network. Device 1000 can be a client computer or a server. As shown in FIG. 10, device 1000 can be any suitable type of41MOFO-359650473Attorney Docket No: 197102019240 microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s) 1010, input devices 1020, output devices 1030, memory or storage devices 1040, communication devices 1060, and nucleic acid sequencers 1070. Software 1050 residing in memory or storage device 1040 may comprise, e.g., an operating system as well as software for executing the methods described herein, e.g., for detecting CN or CN ratio of a gene encoding a cancer-expressed antigen of the disclosure. Input device 1020 and output device 1030 can generally correspond to those described herein, and can either be connectable or integrated with the computer.

[0165] Input device 1020 can be any suitable device that provides input, such as a touch screen, keyboard or keypad, mouse, or voice -recognition device. Output device 1030 can be any suitable device that provides output, such as a touch screen, haptics device, or speaker.

[0166] Storage 1040 can be any suitable device that provides storage (e.g., an electrical, magnetic or optical memory including a RAM (volatile and non-volatile), cache, hard drive, or removable storage disk). Communication device 1060 can include any suitable device capable of transmitting and receiving signals over a network, such as a network interface chip or device. The components of the computer can be connected in any suitable manner, such as via a wired media (e.g., a physical system bus 1080, Ethernet connection, or any other wire transfer technology) or wirelessly (e.g., Bluetooth®, Wi-Fi®, or any other wireless technology).

[0167] Software module 1050, which can be stored as executable instructions in storage 1040 and executed by processor(s) 1010, can include, for example, an operating system and / or the processes that embody the functionality of the methods of the present disclosure, e.g., for d predicting outcome of treating an individual having cancer with a treatment comprising an ADC (e.g., as embodied in the devices as described herein).

[0168] Software module 1050 can also be stored and / or transported within any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described herein, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a computer-readable storage medium can be any medium, such as storage 1040, that can contain or store processes for use by or in connection with an instruction execution system, apparatus, or device. Examples of computer-readable storage media may include memory units like hard drives, flash drives and distribute modules that operate as a single functional unit. Also, various processes described herein may be embodied as modules configured to operate in accordance with the embodiments and techniques described above. Further, while processes may be shown and / or described separately, those skilled in the art will appreciate that the above processes may be routines or modules within other processes.42MOFO-359650473Attorney Docket No: 197102019240

[0169] Software module 1050 can also be propagated within any transport medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described above, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a transport medium can be any medium that can communicate, propagate or transport programming for use by or in connection with an instruction execution system, apparatus, or device. The transport readable medium can include, but is not limited to, an electronic, magnetic, optical, electromagnetic or infrared wired or wireless propagation medium.

[0170] Device 1000 may be connected to a network (e.g., network 1104, as shown in FIG. 11 and described below), which can be any suitable type of interconnected communication system. The network can implement any suitable communications protocol and can be secured by any suitable security protocol. The network can comprise network links of any suitable arrangement that can implement the transmission and reception of network signals, such as wireless network connections, T1 or T3 lines, cable networks, DSL, or telephone lines.

[0171] Device 1000 can be implemented using any operating system, e.g., an operating system suitable for operating on the network. Software module 1050 can be written in any suitable programming language, such as C, C++, Java or Python. In various embodiments, application software embodying the functionality of the present disclosure can be deployed in different configurations, such as in a client / server arrangement or through a Web browser as a Web-based application or Web service, for example. In some embodiments, the operating system is executed by one or more processors, e.g., processor(s) 1010.

[0172] Device 1000 can further include a sequencer 1070, which can be any suitable nucleic acid sequencing instrument. Exemplary sequencers can include, without limitation, Roche / 454’s Genome Sequencer (GS) FLX System, Illumina / Solexa’s Genome Analyzer (GA), Illumina’s HiSeq 2500, HiSeq 3000, HiSeq 4000 and NovaSeq 6000 Sequencing Systems, Life / APG’s Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator’s G.007 system, Helicos BioSciences’ HeliScope Gene Sequencing system, or Pacific Biosciences’ PacBio RS system.

[0173] FIG. 11 illustrates an example of a computing system in accordance with one embodiment. In computing system 1100, device 1000 (e.g., as described above and illustrated in FIG. 10) is connected to network 1104, which is also connected to device 1106. In some embodiments, device 1106 is a sequencer. Exemplary sequencers can include, without limitation, Roche / 454’ s Genome Sequencer (GS) FLX System, Illumina / Solexa’s Genome Analyzer (GA), Illumina’s HiSeq 2500, HiSeq 3000, HiSeq 4000 and NovaSeq 6000 Sequencing Systems, Life / APG’s Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator’s G.007 system, Helicos BioSciences’ HeliScope Gene Sequencing system, or Pacific Biosciences’ PacBio RS system.

[0174] Devices 1000 and 1106 may communicate, e.g., using suitable communication interfaces via network 1104, such as a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet.43MOFO-359650473Attorney Docket No: 197102019240In some embodiments, network 1104 can be, for example, the Internet, an intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Devices 1000 and 1106 may communicate, in part or in whole, via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. Additionally, devices 1000 and 1106 may communicate, e.g., using suitable communication interfaces, via a second network, such as a mobile / cellular network. Communication between devices 1000 and 1106 may further include or communicate with various servers such as a mail server, mobile server, media server, telephone server, and the like. In some embodiments, devices 1000 and 1106 can communicate directly (instead of, or in addition to, communicating via network 1104), e.g., via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. In some embodiments, devices 1000 and 1106 communicate via communications 1108, which can be a direct connection or can occur via a network (e.g., network 204).

[0175] One or all of devices 1000 and 1106 generally include logic (e.g., http web server logic) or are programmed to format data, accessed from local or remote databases or other sources of data and content, for providing and / or receiving information via network 1104 according to various examples described herein.

[0176] FIG. 12 illustrates an exemplary process 1200 for predicting outcome of treating an individual having cancer with a treatment comprising an ADC, in accordance with some embodiments of the present disclosure. Process 1200 is performed, for example, using one or more electronic devices implementing a software program. In some examples, process 1200 is performed using a client-server system, and the blocks of process 1200 are divided up in any manner between the server and a client device. In other examples, the blocks of process 1200 are divided up between the server and multiple client devices. Thus, while portions of process 1200 are described herein as being performed by particular devices of a client-server system, it will be appreciated that process 1200 is not so limited. In some embodiments, the executed steps can be executed across many systems, e.g., in a cloud environment. In other examples, process 1200 is performed using only a client device or only multiple client devices. In process 1200, some blocks are, optionally, combined, the order of some blocks is, optionally, changed, and some blocks are, optionally, omitted. In some examples, additional steps may be performed in combination with the process 1200. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

[0177] At block 1202, a plurality of sequence reads of one or more nucleic acid molecules is obtained, wherein the one or more nucleic acid molecules are derived from a sample obtained from an individual, e.g., as described herein. In some embodiments, the sample is obtained from an individual having cancer, such as a cancer described herein. In some embodiments, the sequence reads are obtained using a sequencer, e.g., as described herein or otherwise known in the art. In some embodiments, the nucleic acid molecules comprise one or more nucleic acid molecules corresponding44MOFO-359650473Attorney Docket No: 197102019240 to a gene encoding a cancer-expressed antigen (or fragment thereof). Optionally, prior to obtaining the sequence reads, the sample is purified, enriched (e.g., for nucleic acid(s) corresponding to a gene encoding a cancer-expressed antigen of the disclosure or fragments thereof), and / or subjected to PCR amplification. At block 1204, an exemplary system (e.g., one or more electronic devices) analyzes the plurality of sequence reads for CN or CN ratio of the gene encoding the cancer-expressed antigen, e.g., in the sample. At block 1206, the system detects (e.g., based on the analysis) CN or CN ratio of the gene encoding the cancer-expressed antigen in the sample. Optionally, the system detects or receives input (e.g., acquires a value) corresponding to ploidy of the sample. In some embodiments, at block 1206, the system detects (e.g., based on the analysis and sample ploidy) CN ratio of the gene encoding the cancer-expressed antigen in the sample. At block 1208, the system compares CN ratio of the gene encoding the cancer-expressed antigen (e.g., in the sample) to a threshold CN ratio, e.g., 1, 2, 2.5, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20. In some embodiments, at block 1210, in accordance with a determination that the CN ratio of the gene encoding the cancer-expressed antigen (e.g., in the sample) is greater than or equal to the threshold CN ratio, the system predicts an improved outcome associated with treating the individual with an ADC (e.g., whose antibody component specifically binds the cancer-expressed antigen), e.g., as compared to outcome of treating an individual whose sample has a CN ratio of the gene that is less than the threshold CN ratio with a treatment comprising the ADC. In some embodiments, at block 1212, in accordance with a determination that the CN ratio of the gene encoding the cancer-expressed antigen (e.g., in the sample) is less than the threshold CN ratio, the system predicts no improved outcome (in some embodiments, a worse outcome) associated with treating the individual with an ADC (e.g., whose antibody component specifically binds the cancer-expressed antigen), e.g., as compared to outcome of treating an individual whose sample has a CN ratio of the gene that is greater than or equal to the threshold CN ratio with a treatment comprising the ADC. In some embodiments, at block 1212, in accordance with a determination that the CN ratio of the gene encoding the cancer-expressed antigen (e.g., in the sample) is less than the threshold CN ratio, the system predicts an improved outcome associated with treating the individual with a therapeutic agent other than an ADC (e.g., a standard of care treatment or chemotherapeutic agent, such as one that does not comprise an ADC), e.g., as compared to outcome of treating an individual whose sample has a CN ratio of the gene that is less than the threshold CN ratio with a treatment comprising an ADC.

[0178] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is biliary tract cancer, breast cancer, gastric cancer, colorectal cancer, or lung cancer. In some embodiments, the cancer is advanced or metastatic. In some embodiments, the treatment comprising the ADC is a first-line treatment. In some embodiments, the treatment comprising the ADC is a second-line or higher treatment.45MOFO-359650473Attorney Docket No: 197102019240

[0179] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the plurality of sequence reads is obtained by sequencing nucleic acids obtained from any of the samples described herein, e.g., tissue and / or liquid biopsies, etc. In some embodiments, the sample is obtained from the cancer. In some embodiments, the sample comprises a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some embodiments, the sample is from a tumor biopsy, tumor specimen, or circulating tumor cell. In some embodiments, the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva. In some embodiments, the sample comprises cells and / or nucleic acids from the cancer. In some embodiments, the sample comprises mRNA, DNA, circulating tumor DNA (ctDNA), cell-free DNA, or cell-free RNA from the cancer. In some embodiments, the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs). In some embodiments, the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0180] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the plurality of sequence reads is obtained by sequencing. In some embodiments, the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing technique comprises next generation sequencing (NGS).

[0181] In some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, the disclosed methods for predicting outcome of treating an individual having cancer with a treatment comprising an ADC may be implemented as part of a genomic profiling process that comprises identification of the presence of variant sequences at one or more gene loci in a sample derived from an individual as part of detecting, monitoring, predicting a risk factor, or selecting a treatment for a particular disease, e.g., cancer. In some instances, the variant panel selected for genomic profiling may comprise the detection of variant sequences at a selected set of gene loci. In some instances, the variant panel selected for genomic profiling may comprise detection of variant sequences at a number of gene loci through comprehensive genomic profiling (CGP), a next-generation sequencing (NGS) approach used to assess hundreds of genes (including relevant cancer biomarkers) in a single assay.

[0182] In some instances, a molecular profile may comprise information on the presence of genes (or variant sequences thereof), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’s genome and / or proteome, as well as information on the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors.

[0183] In some instances, a molecular profile for the individual may comprise results from a comprehensive genomic profiling (CGP) test, a nucleic acid sequencing-based test, a gene expression46MOFO-359650473Attorney Docket No: 197102019240 profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof.

[0184] Accordingly, in some embodiments of any of the methods, systems, devices, non-transitory computer readable storage media, or processes of the disclosure, a molecular profile for the sample or for the individual is generated based, at least in part, on detecting CN or CN ratio of a gene encoding a cancer-expressed antigen in the sample. In some embodiments, the individual is administered a treatment based at least in part on the molecular profile, e.g., a treatment described herein such as an ADC. In some embodiments, the molecular profile further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile further comprises results from a nucleic acid sequencingbased test. In some embodiments, the molecular profile further comprises / indicates / comprises information on presence or absence of mutations in one or more additional genes, e.g., a panel of known / suspected oncogenes and / or tumor suppressors. In some embodiments, the molecular profile is obtained from a genomic profiling assay (such as a cancer- or tumor-related genomic profiling assay), e.g., as obtained using any of the sequencing methodologies described herein. In some embodiments, the molecular profile includes information from whole-genome or whole-exome sequencing. In some embodiments, the molecular profile includes information from targeted sequencing. In some embodiments, the molecular profile includes information from NGS. In some embodiments, the molecular profile comprises / indicates / comprises information on presence or absence of mutations such as short variant alterations (e.g., a base substitution, insertion, or deletion), copy-number alterations (e.g., an amplification or a homozygous deletion), and / or rearrangements (e.g., a gene fusion or other genomic or chromosomal rearrangement) of one or more genes, e.g., a panel of known / suspected oncogenes and / or tumor suppressors.IV. Exemplary Embodiments

[0185] The following exemplary embodiments are representative of some aspects of the invention:Embodiment 1. A method of treating or delaying progression of cancer in an individual, comprising:(a) acquiring knowledge of or detecting a copy number (CN) ratio of greater than or equal to 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and(b) administering to the individual a treatment comprising an antibody-drug conjugate (ADC), wherein the antibody of the ADC specifically binds the antigen.47MOFO-359650473Attorney Docket No: 197102019240Embodiment 2. A method of predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the antibody of the ADC specifically binds the antigen, wherein the CN ratio of the antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.Embodiment 3. The method of embodiment 2, wherein the improved outcome comprises longer progression-free survival (PFS), longer overall survival (OS), and / or longer time to treatment discontinuation (TTD).Embodiment 4. The method of any one of embodiments 1-3, wherein the CN ratio of the gene encoding the antigen in the sample is greater than 2.5.Embodiment 5. The method of embodiment 4, wherein the CN ratio of the gene encoding the antigen in the sample is less than or equal to 5.Embodiment 6. The method of any one of embodiments 1-3, wherein the CN ratio of the gene encoding the antigen in the sample is greater than 5.Embodiment 7. The method of embodiment 6, wherein the CN ratio of the gene encoding the antigen in the sample is less than or equal to 10.Embodiment 8. The method of any one of embodiments 1-3, wherein the CN ratio of the gene encoding the antigen in the sample is greater than 10, greater than 15, or greater than 20.Embodiment 9. A method of treating or delaying progression of cancer, comprising:(a) acquiring knowledge of or detecting a copy number (CN) ratio of less than 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from an individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and(b) administering to the individual a therapeutic agent other than an antibody-drug conjugate (ADC).48MOFO-359650473Attorney Docket No: 197102019240Embodiment 10. The method of embodiment 9, wherein the CN ratio of the gene encoding the antigen is less than or equal to 0.5.Embodiment 11. The method of embodiment 9 or embodiment 10, wherein the therapeutic agent other than an ADC comprises a standard of care treatment or a chemotherapeutic agent.Embodiment 12. The method of any one of embodiments 1-11, wherein the ploidy of the sample refers to an average ploidy of two or more segments of the genome.Embodiment 13. The method of any one of embodiments 1-12, wherein ploidy of the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), comparative genomic hybridization (CGH), or microarray analysis.Embodiment 14. The method of any one of embodiments 1-13, wherein CN of the gene encoding the antigen in the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), in situ hybridization (ISH), comparative genomic hybridization (CGH), or microarray analysis.Embodiment 15. The method of embodiment 13 or embodiment 14, wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).Embodiment 16. The method of any one of embodiments 1-15, further comprising obtaining the sample from the individual.Embodiment 17. The method of any one of embodiments 1-16, wherein the sample is obtained from the cancer.Embodiment 18. The method of any one of embodiments 1-17, wherein the sample comprises a tissue biopsy sample or a liquid biopsy sample.Embodiment 19. The method of embodiment 18, wherein the sample is from a tumor biopsy or tumor specimen.49MOFO-359650473Attorney Docket No: 197102019240Embodiment 20. The method of embodiment 18, wherein the sample is a liquid biopsy sample and comprises circulating tumor cells (CTCs).Embodiment 21. The method of embodiment 18, wherein the sample is a liquid biopsy sample and comprises cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.Embodiment 22. The method of embodiment 18, wherein the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva.Embodiment 23. The method of any one of embodiments 1-22, wherein the sample comprises cells and / or nucleic acids from the cancer.Embodiment 24. The method of embodiment 23, wherein the sample comprises DNA, circulating tumor DNA (ctDNA), or cell-free DNA from the cancer.Embodiment 25. The method of any one of embodiments 1-24, wherein the cancer is a solid tumor.Embodiment 26. The method of any one of embodiments 1-25, wherein the cancer is biliary tract cancer, breast cancer, gastric cancer, colorectal cancer, or lung cancer.Embodiment 27. The method of any one of embodiments 1-26, wherein the cancer is advanced or metastatic.Embodiment 28. The method of any one of embodiments 1-8 and 12-27, wherein the treatment comprising the ADC is an adjuvant or neoadjuvant treatment.Embodiment 29. The method of any one of embodiments 1-8 and 12-27, wherein the treatment comprising the ADC is a first-line (IL) treatment.Embodiment 30. The method of any one of embodiments 1-8 and 12-27, wherein the treatment comprising the ADC is a second-line (2L) or higher treatment.Embodiment 31. The method of any one of embodiments 1-30, wherein the antigen expressed by the cancer is expressed on the surface of cells of the cancer.50MOFO-359650473Attorney Docket No: 197102019240Embodiment 32. The method of any one of embodiments 1-31, wherein the antigen expressed by the cancer is HER2, and the antibody of the ADC specifically binds HER2.Embodiment 33. The method of embodiment 32, wherein the ADC is trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), disitamab vedotin (RC48), trastuzumab duocarmazine (SYD985), XMT-1522, ZRC-3256, MRG002, ARX788, B DC-1001, A166, FS-1502, SHR-A1201, DP303c, BI-CON-02, ALT-P7, DX126-262, ZW49, HS630, B003, SBT6050, SHR-A1811, MT-5111, GQ1001, PF-06804103, or T-PNU.Embodiment 34. The method of embodiment 32 or embodiment 33, wherein the cancer expresses HER2 at a level of 3 or higher, as measured by immunohistochemistry (IHC), or wherein the cancer is positive for HER2 expression by in situ hybridization (ISH) and shows HER2 expression at a level of 2 or higher as measured by IHC.Embodiment 35. The method of embodiment 32 or embodiment 33, wherein the cancer expresses HER2 at a level of 0 as measured by IHC, or wherein the cancer is negative for HER2 expression by ISH and shows HER2 expression at a level of 1 or higher or 2 or higher.Embodiment 36. The method of any one of embodiments 32-35, wherein the individual has received prior treatment with a HER2-targeted therapy.Embodiment 37. The method of embodiment 36, wherein the HER2-targeted therapy comprises trastuzumab and / or pertuzumab.Embodiment 38. The method of any one of embodiments 32-35, wherein the individual has not received prior treatment with a HER2-targeted therapy.Embodiment 39. The method of any one of embodiments 1-31, wherein the antigen expressed by the cancer is c-mesenchymal-epithelial transition (c-MET), and the antibody of the ADC specifically binds c-MET.Embodiment 40. The method of embodiment 39, wherein the ADC is telisotuzumab vedotin (Teliso-V, ABBV-399), RC108, or HRA00129-C004.Embodiment 41. The method of any one of embodiments 1-31, wherein the antigen expressed by the cancer is trophoblast cell surface antigen 2 (Trop-2), and the antibody of the ADC specifically binds Trop-2.51MOFO-359650473Attorney Docket No: 197102019240Embodiment 42. The method of embodiment 41, wherein the ADC is sacituzumab govitecan, datopotamab deruxtecan (Dato-DXd), SKB-264, BIO-106, DB-1305, ESG-401, BAT-8008, BL- M02D1, D AC-002 (JS1O8), FDA018, or SHR-A1921.Embodiment 43. The method of any one of embodiments 1-31, wherein the antigen expressed by the cancer is folate receptor alpha (FRa), and the antibody of the ADC specifically binds FRa.Embodiment 44. The method of embodiment 43, wherein the ADC is mirvetuximab soravtansine, luveltamab tazevibulin (STRO-002), or MORAb-202.Embodiment 45. The method of any one of embodiments 1-31, wherein the antigen expressed by the cancer is claudin 18, and the antibody of the ADC specifically binds claudin 18.Embodiment 46. The method of embodiment 45, wherein the ADC is CMG901, IBI343, SKB315 (MK-1200), TORL-2-307-ADC, SYSA1801, AZD0901 (CMG901), ATG-022, SOT102, or EO-3021.Embodiment 47. A system for predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: a memory configured to store one or more program instructions; and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to:(a) obtain a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer;(b) analyze the plurality of sequence reads for copy number of a gene encoding an antigen expressed by the cancer; and(c) calculate, based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples, wherein the CN ratio refers to CN of the gene encoding the antigen in the one or more samples divided by ploidy of the one or more samples; wherein the antibody of the ADC specifically binds the antigen, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.52MOFO-359650473Attorney Docket No: 197102019240Embodiment 48. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), the method comprising:(a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are derived from one or more samples obtained from an individual having a cancer;(b) analyzing, using the one or more processors, the plurality of sequence reads for copy number of a gene encoding an antigen expressed by the cancer; and(c) calculating, using the one or more processors and based on the analyzing, a copy number (CN) ratio of the gene encoding the antigen in the one or more samples, wherein the CN ratio refers to CN of the gene encoding the antigen in the one or more samples divided by ploidy of the one or more samples; wherein the antibody of the ADC specifically binds the antigen, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.

[0186] The method steps of the invention(s) described herein are intended to include any suitable method of causing one or more other parties or entities to perform the steps, unless a different meaning is expressly provided or otherwise clear from the context. Such parties or entities need not be under the direction or control of any other party or entity, and need not be located within a particular jurisdiction. Thus, for example, a description or recitation of "adding a first number to a second number" includes causing one or more parties or entities to add the two numbers together. For example, if person X engages in an arm's length transaction with person Y to add the two numbers, and person Y indeed adds the two numbers, then both persons X and Y perform the step as recited: person Y by virtue of the fact that he actually added the numbers, and person X by virtue of the fact that he caused person Y to add the numbers. Furthermore, if person X is located within the United States and person Y is located outside the United States, then the method is performed in the United States by virtue of person X's participation in causing the step to be performed.

[0187] The terminology used in the description of the various described embodiments herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the description of the various described embodiments and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “includes,” “including,” “comprises,” and / or “comprising,” when53MOFO-359650473Attorney Docket No: 197102019240 used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0188] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. To the extent that any reference incorporated by reference conflicts with the instant disclosure, the instant disclosure shall control.EXAMPLES

[0189] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Example 1: Targeting clinically advanced breast cancer with conjugated and unconjugated HER2 antibodies: Does copy number matter?

[0190] HER2-targeted therapy is broadly used in advanced breast cancer (aBC). For HER2+ aBC (HER2 IHC 3+ or 2+ / ISH+), IL standard of care includes unconjugated HER2 antibodies trastuzumab and pertuzumab (HP) in combination with chemotherapy. In the 2L+ setting, antibody drug conjugate trastuzumab deruxtecan (T-DXd) can be used for HER2 low (IHC 1+, 2+ / ISH-) patients with significant benefit seen in Destiny Breast 04 relative to physicians’ choice chemotherapy. For some targeted therapies, including MET inhibitor capmatinib, the magnitude of genomic copy number (CN) gains predict benefit. Here, a real-world cohort of aBC patients (pts) treated with HER2 antibody therapies was examined to determine if HER2 genomic CN ratio predicts outcomes.Materials and Methods

[0191] This study used a nationwide, de-identified clinico-genomic database including patient-level structured and unstructured retrospective longitudinal clinical and genomic data from approximately 280 cancer clinics (approximately 800 care sites) in the United States and included patients who underwent tissue comprehensive genomic profiling after 2014. HER2 CN ratio was defined as ratio of modeled absolute CN to average specimen ploidy.

[0192] A schematic illustrating the different cohorts analyzed in the study is shown in FIG. 1. These included patients with aBC who are HER2+ (HER2 IHC 3+ or 2+ / ISH+) treated with trastuzumab and pertuzumab (HP) in combination with chemotherapy in first-line (Cohort 1), patients with aBC who54MOFO-359650473Attorney Docket No: 197102019240 have not received prior HER2-targeted therapies and who received treatment with T-DXd in the 2L or 3L setting (Cohort 2), and patients with aBC who are HER2 low / negative (HER2 IHC 0 or 1+ to 2+ / ISH-) that have not received prior HER2-targeted therapies and who received treatment with T- DXd in the 2L or 3L setting (Cohort 3).Results

[0193] 121 HER2+ aBC patients were treated with HP + chemotherapy in IL and received genomic profiling (Cohort 1). HER2 CN ratio was generally high (median HER2 CN ratio of 10; IQR 2.8- 17.8). Pts with a HER2 CN ratio of >5 (equivalent to CN 10 in a diploid tumor) had significantly better TTD (median 8.4 v 5.3 mo, HR = 0.55, 95% CI 0.36-0.84, p = 0.006), and OS (median 76 v 3 Imo, HR = 0.33 95% CI 0.18-0.61, p <0.001) than pts with a HER2 CN ratio of <5 (FIGS. 2A-2C), and comparable trends were observed for PFS (median 13v 9mo, HR = 0.81 (95% CI 0.52-1.26), p = 0.35) (FIGS. 2A-2C). Similar benefit was seen for pts with a HER2 CN ratio of 5-10, 10-15, 15-20, and 20+ relative to HER2 ratio <5. No differences in outcome were observed between patients having CN <2.5 and 2.5-5 (FIGS. 3A-3C). Patients with HER2 CN ratio of greater than 5 showed improved outcomes, as compared to those with CN ratio of less than or equal to 2.5 (FIGS. 4A-4C & 5A-5C). Once the CN ratio was above 5, hazard ratios were similar across different CN levels.

[0194] For Cohort 2 patients, data showed a lack of follow-up time, since T-DXd approval was recent (FIGS. 6A-6C). No difference in outcome was observed between patients with HER2 CN ratio of less than 2.5 and 2.5-5.

[0195] For 95 HER2 low pts treated with T-DXd in the 2E or 3E setting (Cohort 3), HER2 CN ratio was generally low (median ratio of 1, IQR 1-1) with a maximum HER2 CN ratio of 2.5 in the cohort. Across HER2 CN levels, outcomes were similar with the exception of pts with a HER2 CN ratio of <0.5 (11 / 95; 12%) who had significantly worse PFS (median 2.5 v 6.1 mo, HR = 0.37, 95% CI 0.17- 0.79 p = 0.01) and OS (median 6.5 v 25.2 mo; HR =0.32, 95% CI 0.14-0.75; p=0.008) than pts with a HER2 CN ratio of >0.5, with similar trends for TTD (median 1.6 v 4.8 mo, HR = 0.52, 95% CI 0.26- 1.06 p = 0.07). No difference in outcomes was observed between patients with HER2 CN less than 2.5 and 2.5-5 (FIGS. 7A-7C). HER2 low pts with HER2 CN ratio <0.5 were significantly less likely to benefit from T-DXd than patients with HER2 CN ratio >0.5 (FIGS. 8A-8C). Patients with HER2 CN ratio greater than 0.5 had a better outcome than those with CN ratio less than or equal to 0.5 (FIGS. 9A-9C).

[0196] These results show that, in a cohort of real-world aBC pts treated with HER2 antibody therapies, HER2 CN ratio was significantly associated with clinical outcomes. For unconjugated antibodies, pts with a HER2 CN ratio of <5 had significantly shorter TTD, PFS and OS. For the HER2 conjugated antibody T-DXd, pts with HER2 low BC with a hemizygous or deep deletion in HER2 (HER2 CN ratio <0.5) had significantly worse outcomes, consistent with partial or complete55MOFO-359650473Attorney Docket No: 197102019240 target loss, reflecting potential additional predictive value of an NGS-based HER2 CN quantitative biomarker that could be deployed in conjunction with the currently employed qualitative IHC status biomarker to identify pts for whom an alternative therapy may be more efficacious. For pts with a ratio of >0.5, benefit was seen across the range of HER2 CN ratios suggesting that even low levels of HER2 are enough for activity of T-DXd, consistent with the high pay load: antibody ratio and bystander effect for T-DXd.56MOFO-359650473

Claims

Attorney Docket No: 197102019240CLAIMSWhat is claimed is:

1. A method of treating or delaying progression of cancer in an individual, comprising:(a) detecting a copy number (CN) ratio of greater than or equal to 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and(b) administering to the individual a treatment comprising an antibody-drug conjugate (ADC), wherein the antibody of the ADC specifically binds the antigen.

2. A method of treating or delaying progression of cancer, comprising:(a) detecting a copy number (CN) ratio of less than 1 of a gene encoding an antigen expressed by the cancer in a sample obtained from an individual, wherein the CN ratio refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample; and(b) administering to the individual a therapeutic agent other than an antibody-drug conjugate (ADC).

3. The method of claim 2, wherein the therapeutic agent other than an ADC comprises a standard of care treatment or a chemotherapeutic agent.

4. A method of predicting outcome of treating an individual having cancer with a treatment comprising an antibody-drug conjugate (ADC), comprising: acquiring knowledge of or detecting a copy number (CN) ratio of a gene encoding an antigen expressed by the cancer in a sample obtained from the individual, wherein the antibody of the ADC specifically binds the antigen, wherein the CN ratio of the antigen refers to CN of the gene encoding the antigen in the sample divided by ploidy of the sample, and wherein a CN ratio of greater than or equal to 1 of the gene encoding the antigen in the sample predicts an improved outcome associated with treating the individual with a treatment comprising the ADC, as compared to outcome of treating an individual whose sample has a CN ratio of the gene encoding the antigen that is less than 1 with a treatment comprising the ADC.

5. The method of claim 1, wherein the CN ratio of the gene encoding the antigen in the sample is greater than 2.5 but less than or equal to 5.57MOFO-359650473Attorney Docket No: 1971020192406. The method of claim 1 , wherein the CN ratio of the gene encoding the antigen in the sample is:(a) greater than 5 but less than or equal to 10;(b) greater than 10;(c) greater than 15; or(d) greater than 20.

7. The method of claim 1, wherein the ploidy of the sample refers to an average ploidy of two or more segments of the genome; optionally wherein ploidy of the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), comparative genomic hybridization (CGH), or microarray analysis.

8. The method of claim 1, wherein CN of the gene encoding the antigen in the sample is detected by sequencing, real-time polymerase chain reaction (RT-PCR), in situ hybridization (ISH), comparative genomic hybridization (CGH), or microarray analysis; optionally wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and optionally wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

9. The method of claim 1, wherein the sample comprises a tissue biopsy sample or a liquid biopsy sample.

10. The method of claim 1, wherein the cancer is biliary tract cancer, breast cancer, gastric cancer, colorectal cancer, or lung cancer; optionally wherein the cancer is advanced or metastatic.

11. The method of claim 1 , wherein the treatment comprising the ADC is an adjuvant or neoadjuvant treatment.

12. The method of claim 1, wherein the treatment comprising the ADC is a first-line (IL) treatment, second-line (2L) treatment, or higher than 2L treatment.

13. The method of claim 1, wherein the antigen expressed by the cancer is expressed on the surface of cells of the cancer.

14. The method of claim 1, wherein the antigen expressed by the cancer is HER2, and the antibody of the ADC specifically binds HER2; optionally wherein the ADC is trastuzumab emtansine58MOFO-359650473Attorney Docket No: 197102019240(T-DM1), trastuzumab deruxtecan (T-DXd), disitamab vedotin (RC48), trastuzumab duocarmazine (SYD985), XMT-1522, ZRC-3256, MRG002, ARX788, BDC-1001, A166, FS-1502, SHR-A1201, DP303c, BI-CON-02, ALT-P7, DX126-262, ZW49, HS630, B003, SBT6050, SHR-A1811, MT- 5111, GQ 1001, PF-06804103, or T-PNU.

15. The method of claim 14, wherein:(a) the cancer expresses HER2 at a level of 3 or higher, as measured by immunohistochemistry (IHC), or wherein the cancer is positive for HER2 expression by in situ hybridization (ISH) and shows HER2 expression at a level of 2 or higher as measured by IHC; or(b) the cancer expresses HER2 at a level of 0 as measured by IHC, or wherein the cancer is negative for HER2 expression by ISH and shows HER2 expression at a level of 1 or higher or 2 or higher.

16. The method of claim 14, wherein the individual has received prior treatment with a HER2- targeted therapy; optionally wherein the HER2-targeted therapy comprises trastuzumab and / or pertuzumab.

17. The method of claim 1, wherein the antigen expressed by the cancer is c-mesenchymal- epithelial transition (c-MET), and the antibody of the ADC specifically binds c-MET; optionally wherein the ADC is telisotuzumab vedotin (Teliso-V, ABBV-399), RC108, or HRA00129-C004.

18. The method of claim 1, wherein the antigen expressed by the cancer is trophoblast cell surface antigen 2 (Trop-2), and the antibody of the ADC specifically binds Trop-2; optionally wherein the ADC is sacituzumab govitecan, datopotamab deruxtecan (Dato-DXd), SKB-264, BIO-106, DB- 1305, ESG-401, BAT-8008, BL-M02D1, DAC-002 (JS108), FDA018, or SHR-A1921.

19. The method of claim 1, wherein the antigen expressed by the cancer is folate receptor alpha (FRa), and the antibody of the ADC specifically binds FRa; optionally wherein the ADC is mirvetuximab soravtansine, luveltamab tazevibulin (STRG-002), or MORAb-202.

20. The method of claim 1, wherein the antigen expressed by the cancer is claudin 18, and the antibody of the ADC specifically binds claudin 18; optionally wherein the ADC is CMG901, IBI343, SKB315 (MK-1200), TORL-2-307-ADC, SYSA1801, AZD0901 (CMG901), ATG-022, SOT102, or EO-3021.59MOFO-359650473

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