Nectin-4 specific bicyclic conjugates and uses thereof

Abstract

To provide a method for identifying or selecting a patient.SOLUTION: A method for identifying or selecting patients with elevated Nectin-4 proteins and / or RNA expression levels in tumor tissues, comprising measuring Nectin - 4DNA copy number in tumor tissues or circulating tumor DNA of patients, and selecting patients with elevated Nectin - 4DNA copy number in the tumor tissues, wherein in one embodiment, the step of measuring the Nectin - 4DNA copy number in tumor tissues or circulating tumor DNA of patients comprises using next-generation sequencing technology or sequence-based sequence capture.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bicyclic toxin conjugate or bicyclic tumor-targeting immune agonist (TICA) specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and its use for preventing or treating a disease, disorder, or condition characterized by overexpression of Nectin-4 in diseased tissue, for example, tumor tissue. [Background technology]

[0002] The phenomenon of tumor gene amplification associated with upregulation of the associated encoded protein and the use of tests to determine the presence or absence of gene amplification as a predictor of response to therapy are well established in the treatment of solid tumors. ERBB2 (HER2), MET, and FGFR amplifications have all been demonstrated to predict response to therapies targeting these specific proteins {Arnoud et al., Clinical Cancer Research 2007; Cui JJ, J of Med Chem 2014; Pearson et al., Cancer Discovery 2016}. Overexpression of nectin-4 has been reported in multiple tumor types {Challita-Eid et al., Cancer Research 2016} and is associated with underlying nectin-4 gene amplification in breast cancer {N Pavlova et al., Elife 2013}. Analysis of TCGA data indicates that nectin-4 amplification occurs in additional indications and may therefore represent a method to identify tumors with high nectin-4 expression across indications that may respond to nectin-4-targeted therapeutics such as BT8009.

[0003] Cyclic peptides can bind to protein targets with high affinity and target specificity, and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, several cyclic peptides, such as the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide, have already been successfully used in clinics (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The favorable binding properties are due to the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces measuring several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å, Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å) (Xiong et al. (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upin-1 that binds to urokinase-type plasminogen activator (603 Å, Zhao et al. (2007), J Struct Biol 160(1), 1-10).

[0004] Due to their cyclic structure, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss upon target binding and higher binding affinity. This reduced flexibility locks target-specific conformations, improving binding specificity compared to linear peptides. This effect is exemplified by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which lose selectivity over other MMPs when the ring is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The favorable binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0005] Various research teams have previously tethered polypeptides containing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds that mimic the structure of protein surfaces (Timmerman et al. (2005), ChemBioChem). A method for generating candidate drug compounds has been developed by linking cysteine-containing polypeptides to molecular scaffolds such as TATA (1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one) (Heinis et al., Angew Chem, Int Ed. 2014;53:1602-1606).

[0006] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine ​​residues and two random six-amino acid regions (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. [Brief explanation of the drawings]

[0007] [Figure 1] Increased and amplification of Nectin-4 has been shown to be statistically significantly associated with increased gene expression in various cancers, including sarcoma, uterine, uterine, endometrial, pancreatic, lung adenocarcinoma, breast, squamous lung, head and neck, cervix, and bladder. [Figure 2A]Genes adjacent to Nectin-4 on the MSK Impact panel and Foundation Medicine panel are shown. [Figure 2B] The gene name, description, genomic location and distance to nectin-4 for SDHC and DDR2 are shown. [Figure 3A] Figure 1 shows the strong association between Nectin-4 DNA copy number and SDHC DNA copy number (CN) in publicly available TCGA data. The association between SDHC CN status and Nectin-4 CN status is shown for individual tumors by tumor cohort. [Figure 3B] Figure 1 shows the strong association between Nectin-4 DNA copy number and DDR2 DNA copy number in publicly available TCGA data. The association between DDR2 CN status and Nectin-4 CN status is shown for individual tumors by tumor cohort. [Figure 4] Figure 1 shows candidate surrogate markers identified for Nectin-4 protein expression = SDHC (or DDR2) amplification. SDHC amplification can be used as a surrogate marker to identify Nectin-4-high expressing tumors, which is useful for predicting response to Nectin-4 bicyclic toxin conjugates (BTC). [Figure 5A] Figure 1 shows an analysis of membrane Nectin-4 DNA copy number versus protein expression in TNBC samples. The X-axis is Nectin-4 Log2 (CN ratio), and the dashed lines in the two plots indicate the quartiles of H-score and Nectin-4 DNA copy number. [Figure 5B] Figure 1 shows an analysis of Nectin-4 DNA copy number versus protein expression in the cytoplasm of TNBC samples. The X-axis is Nectin-4 Log2 (CN ratio), and the dashed lines in the two plots indicate the quartiles of H-score and Nectin-4 DNA copy number. [Figure 5C]Figure 1 shows an analysis of Nectin-4 DNA copy number versus protein expression in the membrane and cytoplasm in TNBC samples. Note that if the Log2(CN ratio) is ≥ 0.6, all tumor cores have a combined H-score ≥ 100. The X-axis is Nectin-4 Log2(CN ratio), and the dashed lines in the two plots indicate the quartiles of H-score and Nectin-4 DNA copy number. [Figure 5D] A 100% positive predictive value is shown when a tumor membrane + cytoplasmic H score of ≥ 100 is determined using a CN ≥ 3. [Figure 6] (A) Increased copy number of Nectin-4 DNA is associated with a rightward shift in the combined membrane and cytoplasmic H-score. (B) Relationship between H-score cutoff and copy number call determined by the GATK pipeline (https: / / gatk.broadinstitute.org / hc / en-us). (H-score boundaries included.) [Figure 7A] Membrane + cytoplasmic H-score indicates the number of samples meeting the H-score cutoff as the Nectin-4 log2 copy number cutoff increases. Both H-score and log2 CN cutoffs are included. [Figure 7B] The membrane H-score indicates the number of samples meeting the H-score cutoff as the Nectin-4 log2 copy number cutoff increases. Both the H-score and the log2 CN cutoff are included. [Figure 7C] Cytoplasmic H-score indicates the number of samples meeting the H-score cutoff as the Nectin-4 log2 copy number cutoff increases. Both H-score and log2 CN cutoffs are included. [Figure 7D] The % of TNBC samples exceeding distinct membrane + cytoplasmic H-score cutoffs (x-axis) are shown in relation to different Nectin-4 log2 C / N ratio thresholds. [Figure 8A] We show that SDHC can be used as a surrogate for Nectin-4 DNA copy number (and optionally Nectin-4 protein expression). [Figure 8B]We show that DDR2 can be used as a surrogate for Nectin-4 DNA copy number (and optionally Nectin-4 protein expression). [Figure 9] We present a model of the effect of using a Nectin-4 CN cutoff of log2 CN ratio ≥ 0.5 to increase the proportion of patients with higher membrane + cytoplasmic H scores. Methods: Resampling of TNBC TMA membrane + cytoplasmic H scores was performed without replacement using either no log2 CN ratio cutoff, log2 CN ratio ≥ 0, or log2 CN ratio ≥ 0.5, with an expected cohort size of 16 and 10,000 permutations. H score bins are inclusive on the left and exclusive on the right, with the highest bin inclusive on both sides. [Figure 10A] Nectin-4-expressing tumor cells and CD137-expressing immune cells co-localize in human cancers. Transcript co-expression across tumor types in TCGA. [Figure 10B] MultiOmyx™ imaging enables simultaneous investigation of immune infiltration and spatial proteomic profiling within human tumors. A single ROI from a representative HNSCC sample is shown. T cells (CD3+, red), macrophages (CD68+, blue), NK cells (CD56+, green), and tumor cells (PanCK+, cyan) were detected throughout the tumor (top left). Examples of CD137+ CD4+ and CD137+ CD8+ T cells are shown and represented by white and gray arrows, respectively (top right). Co-expression of Nectin-4 (red) and PanCK (blue) on tumor cells (bottom left). Tumor and stromal regions were identified using PanCK and DAPI masks, respectively (bottom right, red and blue, respectively). [Figure 10C] Tumor Nectin-4 expression (left) where total Nectin-4+PanCK+ cells were normalized to total cells, and CD137+ immune infiltrate (right) where total detected CD137+ cells were normalized to total cells. The horizontal line within each box represents the average of the five samples shown. [Figure 10D]Subset analysis of CD137+ immune infiltrates within stromal (left) and tumor (right) regions across samples is shown, including T cells (CD3+ CD4+ and CD3+ CD8+), macrophages (CD68+), NK cells (CD56+), and B cells (CD19+). Data are total cell counts per phenotype normalized to total CD137+ cells detected across samples within each indication. The horizontal line within each box represents the mean of the five samples shown. Summary of the Invention

[0008] As described herein, the inventors discovered a correlation between the level of Nectin-4 protein expression and Nectin-4 DNA copy number in diseased tissues. Nectin-4 is overexpressed in many difficult-to-treat tumors, including NSCLC, TNBC, pancreatic cancer, ovarian cancer, gastric / upper GI cancer, and urothelial carcinoma. Nectin-4 is expressed at relatively low levels in normal adult tissues. Without wishing to be bound by any particular theory or mechanism, tumors with elevated Nectin-4 protein expression and / or elevated Nectin-4 DNA copy number in diseased tissues may be more likely to benefit from treatment with a Nectin-4-specific bicyclic toxin conjugate. In some embodiments, the Nectin-4-specific bicyclic toxin conjugate is BT8009.

[0009] Furthermore, without wishing to be bound by any particular theory or mechanism, tumors with elevated Nectin-4 RNA expression and / or elevated Nectin-4 DNA copy number in diseased tissues may be more likely to benefit from treatment with a Nectin-4-specific bicyclic toxin conjugate. In some embodiments, the Nectin-4-specific bicyclic toxin conjugate is BT8009.

[0010] Furthermore, without wishing to be bound by any particular theory or mechanism, tumors with elevated Nectin-4 protein expression and / or elevated Nectin-4 RNA expression and / or elevated Nectin-4 DNA copy number in diseased tissues that co-localize with CD137-expressing immune cells may be more likely to benefit from treatment with a Nectin-4-specific bicyclic tumor-targeting immune agonist. In some embodiments, the Nectin-4-specific bicyclic toxin conjugate is BT7480.

[0011] Nectin-4 is also known by the following aliases, each of which is equivalent to nectin-4: nectin cell adhesion molecule 4, nectin-4, LNIR, PRR4, poliovirus receptor-related protein 4, poliovirus receptor-related 4, Ig superfamily receptor LNIR, PVRL4, nectin-4, nectin4, and EDSS1.

[0012] In one aspect, the present invention provides a method for identifying or selecting a patient having an elevated Nectin-4 protein level in diseased tissue, the method comprising measuring the Nectin-4 protein level in diseased tissue of the patient, and selecting a patient having an elevated Nectin-4 protein level in diseased tissue.

[0013] In another aspect, provided herein is a method of treating a disease in a patient having elevated Nectin-4 protein levels in diseased tissue, e.g., as determined using the methods described herein, comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0014] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient having elevated Nectin-4 protein levels in diseased tissue, e.g., using a method described herein, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0015] In some embodiments, the disease is cancer, for example, a cancer described herein. In some embodiments, the diseased tissue is tumor tissue. In some embodiments, the bicyclic toxin conjugate specific for Nectin-4 protein is selected from those described herein, for example, BT8009, or a pharmaceutically acceptable salt thereof.

[0016] In another aspect, provided herein is a method of treating a disease in a patient having elevated Nectin-4 protein levels in diseased tissue, e.g., as determined using the methods described herein, comprising administering to the patient a bicyclic tumor-targeting immune agonist specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0017] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient having elevated Nectin-4 protein levels in diseased tissue, e.g., using a method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0018] In some embodiments, the disease is cancer, e.g., a cancer described herein. In some embodiments, the diseased tissue is tumor tissue. In some embodiments, the bicyclic tumor-targeting immune agonist specific for Nectin-4 is selected from those described herein, e.g., BT7480, or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION

[0019] 1. General Description of Certain Embodiments of the Invention: Recently, it has become common for patients with solid tumors to submit tumor tissues for genetic and genomic characterization at the time of initial disease diagnosis or first recurrence using gene panels developed in-house at academic research centers (Cheng et al., J Molecular Diagnostics, 2015; Rothwell et al., Nature Medicine, 2019) or commercially developed (Miller et al., JCO, 2013; Lanman et al., PLoS One, 2015). These panels are typically designed to identify specific somatic mutations, gene fusions, and gene amplification events. In the case of panels that include Nectin-4, Nectin-4 amplification may indicate that tumors tend to express high levels of Nectin-4, thereby identifying patients who may be more likely to respond to BT8009. Furthermore, some panels include genes that are sufficiently close to and typically co-amplified with Nectin-4 on chromosome 1; therefore, detection of SDHC (or DDR2) amplification can act as a surrogate for Nectin-4 amplification, regardless of whether Nectin-4 is included in the panel. Therefore, it is the intention of the present invention to use patient tumor molecular genetic data regarding Nectin-4, SDHC or DDR2 status to increase the proportion of patients with high Nectin-4 expression as determined by IHC who may respond to BT8009.

[0020] SDHC is also known by the following synonyms, all of which are equivalent to SDHC: succinate dehydrogenase complex subunit C; CYB560; succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; succinate dehydrogenase cytochrome B560 subunit, mitochondrial; succinate-ubiquinone oxidoreductase cytochrome B large subunit; succinate dehydrogenase cytochrome B; cytochrome B large subunit; CYBL; SDH3; succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; succinate dehydrogenase complex subunit C integral membrane protein 15 kDa; succinate dehydrogenase integral membrane subunit; succinate dehydrogenase cytochrome B560 subunit; and cytochrome B large subunit of complex II. Integral membrane protein CII-3b; integral membrane protein CII-3; QP-1; PGL3; QPS1; SDHC; CybL; and QPs1.

[0021] DDR2 is also known by the following aliases, each of which is equivalent to DDR2: discoidin domain receptor tyrosine kinase 2; TKT; discoidin domain-containing receptor tyrosine kinase 2; discoidin domain receptor family, member 2; discoidin domain-containing receptor 2; receptor protein tyrosine kinase TKT; CD167 antigen-like family member B; tyrosine protein kinase TYRO10; discoidin domain receptor 2; EC 2.7.10.1; NTRKR3; TYRO10; neurotrophic tyrosine kinase, receptor-related 3; neurotrophic tyrosine kinase receptor-related 3; migration-inducing protein 20; migration-inducing gene 16 protein; hydroxyaryl protein kinase; CD167b antigen; EC 2.7.10; MIG20a; WRCN; and DDR2.

[0022] Next-generation sequencing (NGS), also known as high-throughput sequencing, allows for DNA and RNA sequencing much faster and more inexpensively than previously used Sanger sequencing. In some embodiments, the NGS technology is Illumina (Solexa) sequencing, which identifies DNA bases and adds them to nucleic acid strands simultaneously, with each base emitting a unique fluorescent signal. In some embodiments, the NGS technology is Roche 454 sequencing, which is based on pyrosequencing, a technique that again uses fluorescence to detect pyrophosphate release after a nucleotide is incorporated into a new DNA strand by a polymerase. In some embodiments, the NGS technology is Ion Torrent: Proton / PGM sequencing, which measures the direct release of H+ (protons) from the incorporation of individual bases by a DNA polymerase.

[0023] The Nectin-4, SDHC, and DDR2 DNA copy numbers can be measured by the NGS techniques described herein. In some embodiments, the Nectin-4, SDHC, and / or DDR2 DNA copy numbers are measured by whole genome sequencing. In some embodiments, the Nectin-4, SDHC, and / or DDR2 DNA copy numbers are measured by exome sequencing.

[0024] Array-based sequence capture can also be used to sequence large targeted DNA regions in a time-saving and labor-intensive manner. In some embodiments, an array-based approach is employed to measure Nectin-4, SDHC, and / or DDR2 DNA copy number. In some embodiments, Nectin-4, SDHC, and / or DDR2 DNA copy number is measured using an array-based approach. In some embodiments, the array-based approach is sequence-based. In some embodiments, the array-based approach is non-sequence-based.

[0025] Circulating tumor DNA (ctDNA) refers to DNA found in the bloodstream and originating from cancer cells and tumors. Most DNA is found inside the cell's nucleus. As tumors grow, cells die and are replaced by new ones. These dead cells break down, releasing their contents, including DNA, into the bloodstream. ctDNA is a small piece of DNA, typically composed of less than 200 building blocks (nucleotides) in length. Detecting ctDNA is useful for tumor detection and diagnosis; directing tumor-specific treatment; monitoring treatment; and monitoring symptom-free periods (cancer remission).

[0026] Nectin-4 DNA amplification in tumor tissues has been measured by whole exome sequencing. It has been found that nectin-4 DNA amplification is associated with higher levels of nectin-4 protein expression, and accordingly, may indicate tumors with elevated nectin-4 expression. Without wishing to be bound by any particular theory or mechanism, the present inventors have determined that tumors with elevated nectin-4 DNA copy numbers in diseased tissues may be more likely to benefit from treatment with nectin-4-specific bicyclic toxin conjugates.

[0027] Furthermore, without wishing to be bound by any particular theory or mechanism, the present inventors have determined that tumors with elevated Nectin-4 DNA copy numbers in diseased tissues may be more likely to benefit from treatment with bicyclic tumor-targeting immune agonists specific for Nectin-4.

[0028] SDHC or DDR2 DNA amplification in tumor tissues has been measured by whole exome sequencing.It has been found that SDHC or DDR2 DNA amplification acts as a surrogate for Nectin-4 expression and accordingly indicates tumors with elevated Nectin-4 expression.Without wishing to be bound by any particular theory or mechanism, tumors with elevated SDHC or DDR2 DNA copy numbers in diseased tissues may be more likely to benefit from treatment with Nectin-4-specific bicyclic toxin conjugates.In some embodiments, the Nectin-4-specific bicyclic toxin conjugate is BT8009.

[0029] Furthermore, without wishing to be bound by any particular theory or mechanism, tumors with elevated SDHC or DDR2 DNA copy numbers in diseased tissues may be more likely to benefit from treatment with a Nectin-4-specific bicyclic tumor-targeting immune agonist. In some embodiments, the Nectin-4-specific bicyclic tumor-targeting immune agonist is BT7480.

[0030] Thus, in one aspect, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring the Nectin-4 protein level in the patient's diseased tissue, and selecting a patient having elevated Nectin-4 protein levels in the diseased tissue.

[0031] In some embodiments, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring Nectin-4 mRNA levels in the patient's diseased tissue, and selecting a patient having elevated Nectin-4 mRNA levels in the diseased tissue.

[0032] For each tissue microarray (TMA) core analyzed, the membrane H-score (maximum 300) is added to the cytoplasmic H-score (maximum 300), resulting in a maximum total H-score of 600. This approach differs from the standard approach of calculating the H-score for the entire sample (TMA core), which includes both membrane and cytoplasmic staining, resulting in a maximum H-score of 300. Because the H-score is calculated by % positivity x intensity, cytoplasm is weighted higher than membrane, given its larger surface area, when using the standard approach. Without being bound by any particular theory, one advantage of adding membrane and cytoplasmic H-scores is that it gives equal weight to membrane and cytoplasmic staining (even though membrane surface area is smaller than cytoplasmic surface area), thereby providing a more appropriate score for nectin-4 BTCs, such as BT8009, which are expected to bind membrane nectin-4. Figure 5A shows an analysis comparing isolated membrane H-scores to nectin-4 DNA copy number. Figure 5B shows an analysis of isolated cytoplasmic H-scores versus nectin-4 DNA copy number. Figure 5C shows an analysis of Nectin-4 DNA copy number versus membrane and cytoplasmic protein expression in TNBC samples. Figure 5D shows the 100% positive predictive value when a CN≧3 is used to determine a tumor membrane + cytoplasmic H-score≧100. As shown in Figure 5D, utilizing a cutoff of CN≧3 yields the following values ​​for sensitivity, specificity, positive predictive value, and negative predictive value: Sensitivity = 28.21% (22 / 78) ·Specificity=100%(22 / 22) Positive predictive value = 100% (22 / 22) Negative predictive value = 28.21% (22 / 78) Additionally, Figure 7B (isolated membrane H-score) and Figure 7C (isolated cytoplasmic H-score) show the number of samples meeting the H-score cutoff as the Nectin-4 log2 copy number cutoff increases.

[0033] In some embodiments, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring the Nectin-4 DNA copy number in the patient's diseased tissue, and selecting a patient having elevated Nectin-4 DNA copy number in the diseased tissue.

[0034] In some embodiments, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring SDHC and / or DDR2 DNA copy number in the patient's diseased tissue, and selecting a patient having elevated SDHC and / or DDR2 DNA copy number in the diseased tissue.

[0035] In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 2 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 3 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 4 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 5 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 6 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 7 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 8 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 9 or more. In some embodiments, an elevated level of nectin-4 DNA copy number means that the copy number is 10 or more.

[0036] In some embodiments, a Nectin-4 DNA copy number of 2 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 3 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 4 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 5 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 6 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 7 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 8 or more correlates with an H score of 100 or greater in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 9 or more correlates with an H-score of 100 or more in a Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 10 or more correlates with an H-score of 100 or more in a Nectin-4 IHC staining assay.

[0037] In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 2 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 3 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 4 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 5 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 6 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 7 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 8 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 9 or greater. In some embodiments, an elevated level of SDHC DNA copy number means that the copy number is 10 or greater.

[0038] In some embodiments, an SDHC DNA copy number of 2 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 3 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 4 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 5 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 6 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 7 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 8 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 9 or more correlates with an H score of 100 or greater in an SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 10 or greater correlates with an H score of 100 or greater in an SDHC IHC staining assay.

[0039] In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 2 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 3 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 4 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 5 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 6 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 7 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 8 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 9 or greater. In some embodiments, an elevated level of DDR2 DNA copy number means that the copy number is 10 or greater.

[0040] In some embodiments, a DDR2 DNA copy number of 2 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 3 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 4 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 5 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 6 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 7 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 8 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 9 or more correlates with an H score of 100 or greater in a DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 10 or greater correlates with an H score of 100 or greater in a DDR2 IHC staining assay.

[0041] In another aspect, provided herein is a method for treating a disease in a patient having elevated levels of Nectin-4 protein in diseased tissue, e.g., identified as having elevated levels of Nectin-4 protein in diseased tissue using the methods described herein, comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0042] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient with elevated Nectin-4 protein levels in diseased tissue, e.g., identified as having elevated Nectin-4 protein levels in diseased tissue using a method described herein, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0043] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient in whom Nectin-4 mRNA level, Nectin-4 DNA copy number, SDHC DNA copy number, and / or DDR2 DNA copy number in diseased tissue is elevated, e.g., using a method described herein, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0044] In another aspect, provided herein is a method of treating a disease in a patient with elevated levels of Nectin-4 protein in diseased tissue, e.g., identified as having elevated levels of Nectin-4 protein in diseased tissue using the methods described herein, comprising administering to the patient a bicyclic tumor-targeting immune agonist specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0045] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient with elevated Nectin-4 protein levels in diseased tissue, e.g., identified as having elevated Nectin-4 protein levels in diseased tissue using a method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0046] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient in whom Nectin-4 mRNA level, Nectin-4 DNA copy number, SDHC DNA copy number, and / or DDR2 DNA copy number in diseased tissue is elevated, e.g., using a method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0047] In some embodiments, elevated Nectin-4, SDHC, or DDR2 DNA copy numbers are used to identify patients likely to have tumors with higher Nectin-4 protein expression (measured by IHC). Figure 7D shows that the red curve (log2 C / N ratio ≥ 0.6) is shifted to the right compared to the blue curve (no copy number cutoff). This indicates that Nectin-4 / SDHC / DDR2 amplification can be used to identify and increase the proportion of patients with high Nectin-4 H-scores. Without being bound by any particular theory, such identification would increase the percentage of patients with higher Nectin-4 protein expression (as determined by IHC). This could potentially reduce the frequency of screening failures, defined as (the number of patients not meeting the required H-score cutoff) / (the total number of patients screened).

[0048] This concept is also illustrated in the modeling shown in Figure 9, which shows that using a log2 CN ratio of 0.5 or greater increases the proportion of tumors with higher Nectin-4 protein expression compared to not using this log2 CN ratio cutoff (shown as an all-comers plot). This shift can be seen by comparing the leftmost H-score bins (0–100) between the overall plot (left) and the log2 CN ratio ≥ 0.5 plot (right).

[0049] CD137 (4-1BB) is an immune costimulatory receptor with high therapeutic potential in cancer. After disappointing initial clinical outcomes with agonistic anti-CD137 antibodies, a new generation of systemically targeted CD137 agonists has entered clinical development. Tumor-targeted CD137 agonists were developed using a novel chemical approach based on fully synthetic constrained bicyclic peptide technology. Nectin-4 is a cell adhesion protein overexpressed in multiple human cancers that may benefit from CD137 agonism. BT7480 is the first novel Nectin-4 / CD137 tumor-targeted immune cell agonist (TICA).

[0050] It is well known that several major solid tumor types express Nectin-4 and may be infiltrated by immune cells to varying degrees. Compounds that activate CD137 only when co-bound with Nectin-4 require the appropriate immune-tumor cell proximity. Analysis of RNA expression data from human tumor samples within The Cancer Genome Atlas (TCGA) demonstrated that Nectin-4 and CD137 are co-expressed across several tumor types, including lung, breast, esophagus, stomach, ovary, head and neck, pancreas, and bladder (see Figure 10A). More than half of the tumor types examined had a substantial proportion of tumors expressing both Nectin-4 and CD137 at high levels (see Figure 10A). Based on these data, we selected three major tumor types, namely, NSCLC, HNSCC, and bladder cancer, to further examine Nectin-4 and CD137 in human tumors by spatial proteomic profiling and image analysis (see Example 3).

[0051] Nectin-4 protein levels in tumor tissues were measured by multiplexed immunofluorescence (mIF) assay. Nectin-4 protein levels on tumor cell membranes and in tumor cell cytoplasm have been shown to indicate tumor responsiveness to treatment with Nectin-4-specific bicyclic tumor-targeting immune agonists (TICAs). Without wishing to be bound by any particular theory or mechanism, the present inventors discovered that tumors with elevated Nectin-4 protein levels in lesional tissues are more likely to benefit from treatment with Nectin-4-specific bicyclic tumor-targeting immune agonists (TICAs). It was also found that tumors with elevated Nectin-4 protein levels on tumor cell membranes are more likely to benefit from treatment with BT7480.

[0052] Thus, in one aspect, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein and / or RNA expression levels in diseased tissue, the method comprising measuring Nectin-4 protein and / or RNA expression levels in the patient's diseased tissue, and selecting a patient having elevated Nectin-4 protein and / or RNA expression levels in the diseased tissue.

[0053] In another aspect, provided herein is a method of treating a disease in a patient in which Nectin-4 protein levels and / or RNA expression levels are elevated in diseased tissue, e.g., as determined using the methods described herein, comprising administering to the patient a bicyclic tumor-targeting immune agonist (TICA) specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0054] In another aspect, the present invention provides a method of treating a disease in a patient, comprising selecting a patient having elevated Nectin-4 protein levels and / or RNA expression levels in diseased tissue, e.g., using a method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist (TICA) specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0055] 2. Compounds and Definitions: As used herein, the term "nectin-4-specific bicyclic toxin conjugate" refers to a bicyclic toxin conjugate that specifically binds to nectin-4. Various nectin-4-specific bicyclic toxin conjugates have been previously described, for example, in US2019 / 03889906, WO2019 / 243832, and WO2019 / 243833, the contents of each of which are incorporated herein by reference in their entireties. BT8009 is referred to as BCY8245 in US2019 / 03889906, WO2019 / 243832, and WO2019 / 243833.

[0056] As used herein, the term "BT8009" refers to a bicyclic toxin conjugate having the structure shown below, or a pharmaceutically acceptable salt thereof, wherein the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and the peptide ligand has the amino acid sequence: (β-Ala)-Sar10-CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 1) wherein Sar is sarcosine, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, and Ci, Cii and Ciii represent the first, second and third cysteine ​​residues. [ka] [ka]

[0057] As used herein, the term "Nectin-4-specific bicyclic tumor-targeting immune agonist (TICA)" refers to a bicyclic tumor-targeting immune agonist (TICA) that specifically binds to Nectin-4. Various Nectin-4-specific bicyclic tumor-targeting immune agonists (TICA) have been previously described, for example, in US2019 / 0307836, WO2019 / 193328, US2021 / 0040154, WO2021 / 019244, and WO2021 / 019246, the contents of each of which are incorporated herein by reference in their entirety. BT7480 is referred to as BCY11863 in US2021 / 0040154, WO2021 / 019244, and WO2021 / 019246. The term "BT7480" refers to a bicyclic tumor-targeting immune agonist (TICA), a heterotandem bicyclic peptide conjugate consisting of a nectin-4-specific peptide linked to two CD137-specific peptides via N-(acid-PEG3)-N-bis(PEG3-azide) linkers, and has the structure shown below. [ka]

[0058] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Examples of the salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0059] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. Salt forms are within the scope of the present invention, and it will be understood that a reference to a peptide ligand includes the salt form of the ligand.

[0060] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with the appropriate base or acid in water or an organic solvent, or a mixture of both.

[0061] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Furthermore, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures containing the replacement of hydrogen with deuterium or tritium, or the replacement of a carbon with a C- or C-enriched carbon are within the scope of the invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as therapeutic agents according to the invention.

[0062] As used herein, the term "about" or "approximately" means within 20% of a given value or range. In some embodiments, the term "about" refers to within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value.

[0063] 3. Description of Exemplary Embodiments of the Invention In one aspect, the present invention provides a method for identifying or selecting a patient having an elevated Nectin-4 protein level in diseased tissue, the method comprising measuring the Nectin-4 protein level in the patient's diseased tissue and selecting the patient having an elevated Nectin-4 protein level in the diseased tissue. The Nectin-4 protein level in the diseased tissue can be measured in several ways.

[0064] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, the method comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, and selecting patients who stain positive in the Nectin-4 IHC staining assay.

[0065] As used herein, the term "patients with positive staining" refers to patients in which a certain percentage of cells in tumor tissue sections stain positive in a Nectin-4 IHC staining assay. In some embodiments, a patient with positive staining has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of cells in tumor tissue sections stain positive in a Nectin-4 IHC staining assay.

[0066] There are various methods for measuring staining intensity in IHC staining assays. In some embodiments, staining intensity is measured by visual scoring, e.g., manual scoring using conventional light microscopy. In some embodiments, staining intensity is measured by computed tissue analysis (CTA) scoring. Staining intensity levels can be either no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+). In some embodiments, staining intensity is measured on tumor cell membranes in tumor tissue sections. In some embodiments, staining intensity is measured in tumor cell cytoplasm in tumor tissue sections. In some embodiments, staining intensity is measured in both tumor cell membranes and tumor cell cytoplasm in tumor tissue sections.

[0067] In some embodiments, positive staining refers to an H-score of about 15 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 20 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 30 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 40 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 50 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 75 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 100 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 125 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 150 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 200 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 300 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 400 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of about 500 or greater in tumor tissue sections in an IHC staining assay.

[0068] The H score is the sum of the product of the percentage of cells times the staining intensity of the cells on a 0-3 scale (no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+)) as described above: [((0 × (0 cells%)) + ((1 × (1+ cells%)) + ((2 × (2+ cells%)) + ((3 × (3 cells%))]

[0069] H-scores can be generated for different sections of a tumor tissue section, including, for example, tumor cell membranes and cytoplasm. In some embodiments, the H-score refers to the tumor cell membrane H-score, which is the sum of the products of the percentage of cells times their membrane staining intensity on a scale of 0 to 3, as described above. In some embodiments, the H-score refers to the tumor cell cytoplasm H-score, which is the sum of the products of the percentage of cells times their cytoplasmic staining intensity on a scale of 0 to 3, as described above.

[0070] In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 15 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 20 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 30 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 40 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 50 or greater in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 75 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 100 or greater in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 125 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 150 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 200 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 300 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 400 or greater in tumor tissue sections in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell membranes of about 500 or greater in tumor tissue sections in an IHC staining assay.

[0071] In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 15 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 20 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 30 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 40 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 50 or greater in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 75 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 100 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 125 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 150 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 200 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 300 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 400 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 500 or greater in a tumor tissue section in an IHC staining assay.

[0072] In some embodiments, positive staining refers to a combined H score of tumor cell membranes and tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 15 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 20 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 30 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 40 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 50 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 75 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 100 or greater. In some embodiments, positive staining refers to a combined H score of tumor cell cytoplasm in a tumor tissue section in an IHC staining assay of about 125 or greater. In some embodiments, positive staining refers to a combined H-score of tumor cell cytoplasm of about 150 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to a combined H-score of tumor cell cytoplasm of about 200 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to a combined H-score of tumor cell cytoplasm of about 300 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to a combined H-score of tumor cell cytoplasm of about 400 or greater in a tumor tissue section in an IHC staining assay. In some embodiments, positive staining refers to a combined H-score of tumor cell cytoplasm of about 500 or greater in a tumor tissue section in an IHC staining assay.

[0073] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, the method comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, and selecting patients with an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more.

[0074] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, and selecting patients with a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more.

[0075] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, the method comprising measuring staining intensity in tumor tissue sections of the patients using a Nectin-4 IHC staining assay, and selecting patients with a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more.

[0076] In some embodiments, the present invention provides a method for treating cancer in a patient with elevated Nectin-4 protein levels in tumor tissue, comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0077] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient having elevated Nectin-4 protein levels in tumor tissue, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0078] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring Nectin-4 protein levels in tumor tissue sections of the patient, selecting a patient with elevated Nectin-4 protein levels in the tumor tissue, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0079] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is ovarian cancer.

[0080] In some embodiments, the present invention provides a method of treating cancer in a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the Nectin-4 IHC staining assay is as described herein.

[0081] In some embodiments, the present invention provides a method for treating cancer in a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, the method comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0082] In some embodiments, the present invention provides a method for treating cancer in a patient having a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, the method comprising administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0083] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0084] In some embodiments, the present invention provides a method for treating cancer in a patient, comprising selecting a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0085] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient having a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more in a tumor tissue section in a Nectin-4 IHC staining assay, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0086] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in a tumor tissue section of the patient using a Nectin-4 IHC staining assay; selecting patients with an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more; and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0087] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in a tumor tissue section of the patient using a Nectin-4 IHC staining assay; selecting patients with a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more; and administering a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to the patient.

[0088] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in a tumor tissue section of the patient using a Nectin-4 IHC staining assay; selecting patients with a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, or about 200 or more, or about 300 or more, or about 400 or more, or about 500 or more; and administering a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to the patient.

[0089] In some embodiments, measuring Nectin-4 protein levels in diseased tissues can be performed by measuring Nectin-4 mRNA levels in diseased tissues.

[0090] In some embodiments, measuring Nectin-4 protein levels in diseased tissues is performed using tests commonly used in clinical practice. In some embodiments, measuring Nectin-4 protein levels in diseased tissues is performed by measuring the DNA copy number of other biomarkers, such as Nectin-4, SDHC, and / or DDR2, as described herein. In some embodiments, measuring Nectin-4 protein levels in diseased tissues can be performed by measuring the Nectin-4 DNA copy number in diseased tissues. In some embodiments, measuring Nectin-4 protein levels in diseased tissues can be performed by measuring the SDHC DNA copy number in diseased tissues. In some embodiments, measuring Nectin-4 protein levels in diseased tissues can be performed by measuring the DDR2 DNA copy number in diseased tissues.

[0091] In some embodiments, the present invention provides a method for identifying or selecting a patient with elevated Nectin-4 protein levels in tumor tissue, comprising measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) and selecting a patient with elevated Nectin-4 DNA copy number in the tumor tissue. In some embodiments, the present invention provides a method for identifying or selecting a patient with elevated Nectin-4 protein levels in tumor tissue, comprising measuring the SDHC DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) and selecting a patient with elevated SDHC DNA copy number in the tumor tissue. In some embodiments, the present invention provides a method for identifying or selecting a patient with elevated Nectin-4 protein levels in tumor tissue, comprising measuring the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) and selecting a patient with elevated DDR2 DNA copy number in the tumor tissue. In some embodiments, the method further comprises administering a bicyclic toxin conjugate specific for Nectin-4, such as BT8009, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient having elevated Nectin-4 protein levels in tumor tissue.

[0092] In some embodiments, the patient is a patient with pancreatic cancer. In some embodiments, the patient is a patient with gastric cancer. In some embodiments, the patient is a patient with bladder cancer. In some embodiments, the patient is a patient with head and neck cancer. In some embodiments, the patient is a patient with non-small cell lung cancer (NSCLC). In some embodiments, the patient is a patient with triple-negative breast cancer (TNBC). In some embodiments, the patient is a patient with ovarian cancer.

[0093] In some embodiments, the tumor tissue is lung tumor tissue. In some embodiments, the tumor tissue is ovarian tumor tissue. In some embodiments, the tumor tissue is breast tumor tissue. In some embodiments, the tumor tissue is gastric / upper gastrointestinal (GI) tumor tissue. In some embodiments, the tumor tissue is pancreatic tumor tissue. In some embodiments, the tumor tissue is urothelial tumor tissue. In some embodiments, the tumor tissue is non-small cell lung cancer (NSCLC) tumor tissue. In some embodiments, the tumor tissue is triple-negative breast cancer (TNBC) tumor tissue.

[0094] As used herein, the terms "increase," "elevate," or "enhance" are used interchangeably and encompass any measurable increase in biological function and / or biological activity and / or concentration. For example, the increase can be at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 25-fold, about 50-fold, about 100-fold, or more, compared to a control or baseline amount of function, activity, or concentration.

[0095] As used herein, the term "quantitative analysis of a substance in a sample (e.g., Nectin-4 protein, Nectin-4 mRNA, Nectin-4 DNA copy number, SDHC DNA copy number, or DDR2)" refers to the measurement of a substance in a sample (e.g., Nectin-4 protein, Nectin-4 mRNA, Nectin-4 DNA copy number, SDHC DNA copy number, or DDR2) in a sample. The term "elevated level" of a gene (e.g., DNA copy number) refers to an increase in the amount of a substance of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 25-fold, about 50-fold, about 100-fold, or more, as compared to the amount of the substance in one or more control samples, such as an individual or population of individuals not afflicted with a disease or disorder (e.g., cancer) or an internal control, as determined by techniques known in the art. A subject may also be determined to have an "elevated level" of a substance if the concentration of the substance is increased by one, two, three, four, five, or more standard deviations compared to the mean (average) or median of the substance in a control group of samples, a baseline group of samples, or a retrospective analysis of patient samples. As practiced in the art, such control or baseline levels can be predetermined, measured prior to measurement in the sample, or obtained from a database of such control samples. In other words, the control and subject samples need not be tested simultaneously. In some embodiments, elevated Nectin-4 DNA copy number is measured by Nectin-4 amplification. In some embodiments, elevated SDHC DNA copy number is measured by SDHC amplification. In some embodiments, elevated DDR2 DNA copy number is measured by DDR2 amplification.

[0096] As used herein, the term "nectin-4 amplification" refers to an increase in nectin-4 DNA copy number over normal tissue. In some embodiments, this is expressed as the Log2 of the copy number ratio or nectin-4 Log2 (CN ratio). In some embodiments, the nectin-4 Log2 (CN ratio) is >0.1. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.2. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.3. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.4. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.5. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.6. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.7. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.8. In some embodiments, the nectin-4 Log2 (CN ratio) is >0.9. In some embodiments, the Nectin-4 Log2 (CN ratio) is >1.0.

[0097] As used herein, the term "SDHC amplification" refers to an increase in SDHC DNA copy number over normal tissue. In some embodiments, this is expressed as the Log2 of the copy number ratio or SDHC Log2(CN ratio). In some embodiments, the SDHC Log2(CN ratio) is >0.1. In some embodiments, the SDHC Log2(CN ratio) is >0.2. In some embodiments, the SDHC Log2(CN ratio) is >0.3. In some embodiments, the SDHC Log2(CN ratio) is >0.4. In some embodiments, the SDHC Log2(CN ratio) is >0.5. In some embodiments, the SDHC Log2(CN ratio) is >0.6. In some embodiments, the SDHC Log2(CN ratio) is >0.7. In some embodiments, the SDHC Log2(CN ratio) is >0.8. In some embodiments, the SDHC Log2(CN ratio) is >0.9. In some embodiments, the SDHC Log2(CN ratio) is >1.0.

[0098] As used herein, the term "DDR2 amplification" refers to an increase in DDR2 DNA copy number over normal tissue. In some embodiments, this is expressed as the Log2 of the copy number ratio or the DDR2 Log2 (CN ratio). In some embodiments, the DDR2 Log2 (CN ratio) is >0.1. In some embodiments, the DDR2 Log2 (CN ratio) is >0.2. In some embodiments, the DDR2 Log2 (CN ratio) is >0.3. In some embodiments, the DDR2 Log2 (CN ratio) is >0.4. In some embodiments, the DDR2 Log2 (CN ratio) is >0.5. In some embodiments, the DDR2 Log2 (CN ratio) is >0.6. In some embodiments, the DDR2 Log2 (CN ratio) is >0.7. In some embodiments, the DDR2 Log2 (CN ratio) is >0.8. In some embodiments, the DDR2 Log2 (CN ratio) is >0.9. In some embodiments, the DDR2 Log2 (CN ratio) is >1.0.

[0099] There are various methods for measuring the DNA copy number of Nectin-4, SDHC, and DDR2 in tissues. In some embodiments, the method for measuring the DNA copy number of Nectin-4, SDHC, or DDR2 in a patient's tumor tissue or circulating tumor DNA (ctDNA) comprises using whole exome sequencing.

[0100] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissues, the method comprising measuring Nectin-4 DNA copy number and selecting patients with elevated Nectin-4 DNA copy number.

[0101] In some embodiments, the present invention provides a method for identifying or selecting a patient having an elevated Nectin-4 protein level in tumor tissue, the method comprising measuring SDHC DNA copy number and selecting a patient having an elevated SDHC DNA copy number.

[0102] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissues, comprising measuring DDR2 DNA copy number and selecting patients with elevated DDR2 DNA copy number.

[0103] In some embodiments, the present invention provides methods for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, the method comprising measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) sections using whole exome sequencing, and selecting patients with a Nectin-4 Log2 (CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more.

[0104] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring the SDHC DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) sections using whole exome sequencing, and selecting patients with an SDHC Log2(CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more.

[0105] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) sections using whole exome sequencing, and selecting patients with a DDR2 Log2(CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more.

[0106] In some embodiments, the present invention provides a method of treating cancer in a patient identified as having elevated Nectin-4 protein levels in tumor tissue, e.g., elevated Nectin-4 protein levels in diseased tissue, using the methods described herein, comprising administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0107] In some embodiments, the present invention provides methods of treating cancer in a patient, comprising selecting a patient with elevated Nectin-4 protein levels in tumor tissue, e.g., identified as having elevated Nectin-4 protein levels in diseased tissue using, for example, a method described herein, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0108] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring Nectin-4 protein levels in tumor tissue sections from the patient, e.g., selecting a patient with elevated Nectin-4 protein levels in tumor tissue, identified as having elevated Nectin-4 protein levels in diseased tissue using a method described herein, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein levels are as described herein.

[0109] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is ovarian cancer.

[0110] In some embodiments, the bicyclic toxin conjugate specific for Nectin-4 is selected from compounds described in US2019 / 03889906, WO2019 / 243832, and WO2019 / 243833, each of which is incorporated herein by reference in its entirety.

[0111] In some embodiments, the bicyclic toxin conjugate specific for Nectin-4 is BT8009, as described herein, or a pharmaceutically acceptable salt thereof.

[0112] In some embodiments, the present invention provides a method for treating cancer in a patient having elevated Nectin-4 protein levels in tumor tissue, comprising administering BT8009, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0113] In some embodiments, the present invention provides a method for treating cancer in a patient, comprising selecting a patient having elevated Nectin-4 protein levels in tumor tissue, and administering BT8009, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0114] In some embodiments, the present invention provides a method for treating cancer in a patient, the method comprising measuring Nectin-4 protein levels in tumor tissue of the patient, selecting a patient having elevated Nectin-4 protein levels in tumor tissue, and administering BT8009, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0115] The Nectin-4 specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be administered to a patient in a variety of dosage ranges.

[0116] In some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof at a dose of about 1 mg / kg or less, hi some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof at a dose of about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, or about 0.1 mg / kg.

[0117] In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 1-27 mg / m2. In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 2-20 mg / m2. In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 2.2, 4.4, 7.3, 11, 14.6, or 19.4 mg / m2. In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 2.5, 5.0, 7.5, 10.0, 13.0, or 17.0 mg / m2. In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 1.5-3.5, 3.5-5.5, 6.5-8.5, 10-12, 13.5-15.5, or 18.5-20.5 mg / m2. In some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 1-10 or 10-20 mg / m2, hi some embodiments, the pharmaceutical compositions of the present invention are administered at a dose of about 21, 22, 23, 24, 25, 26, or 27 mg / m2.

[0118] A Nectin-4-specific bicyclic toxin conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof can be administered to a patient at various administration frequencies. In some embodiments, the method of the present invention comprises administering a Nectin-4-specific bicyclic toxin conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a patient at an administration frequency of once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In some embodiments, the method of the present invention comprises administering a Nectin-4-specific bicyclic toxin conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a patient at an administration frequency of twice weekly, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[0119] In some embodiments, the present invention provides a method of treating cancer in a patient with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein and / or RNA expression levels are as described herein.

[0120] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein and / or RNA expression levels are as described herein.

[0121] In some embodiments, the present invention provides a method for identifying or selecting a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, the method comprising measuring Nectin-4 protein and / or RNA expression levels in the patient's tumor tissue and selecting a patient having elevated Nectin-4 protein and / or RNA expression levels in the tumor tissue. In some embodiments, the method further comprises administering a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue.

[0122] In some embodiments, the patient is a patient with pancreatic cancer. In some embodiments, the patient is a patient with gastric cancer. In some embodiments, the patient is a patient with bladder cancer. In some embodiments, the patient is a patient with head and neck cancer. In some embodiments, the patient is a patient with non-small cell lung cancer (NSCLC). In some embodiments, the patient is a patient with triple-negative breast cancer (TNBC). In some embodiments, the patient is a patient with ovarian cancer.

[0123] In some embodiments, the tumor tissue is pancreatic tumor tissue. In some embodiments, the tumor tissue is gastric tumor tissue. In some embodiments, the tumor tissue is bladder tumor tissue. In some embodiments, the tumor tissue is head and neck tumor tissue. In some embodiments, the tumor tissue is non-small cell lung cancer (NSCLC) tumor tissue. In some embodiments, the tumor tissue is triple-negative breast cancer (TNBC) tumor tissue. In some embodiments, the tumor tissue is ovarian tumor tissue.

[0124] As used herein, the term "elevated Nectin-4 protein levels" refers to a certain percentage of cells in tumor tissue having detectable amounts of Nectin-4 protein, for example, on the tumor cell membrane, or in the tumor cell cytoplasm, or both. In some embodiments, Nectin-4 positivity refers to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of cells in tumor tissue having detectable amounts of Nectin-4 protein, for example, on the tumor cell membrane, or in the tumor cell cytoplasm, or both.

[0125] There are various methods for measuring the amount of nectin-4 protein in tissue. In some embodiments, a method for measuring nectin-4 protein levels in a patient's tumor tissue comprises using a nectin-4 multiplexed immunofluorescence (mIF) assay. In some embodiments, the nectin-4 mIF assay comprises staining tumor tissue sections using a rabbit monoclonal α-nectin-4 primary antibody. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody selectively binds to the extracellular domain (ECD) of nectin-4. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody selectively binds to the ECD of nectin-4 is rabbit monoclonal α-nectin-4 primary antibody YMW-1-58.

[0126] In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of up to about 50 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of up to about 40 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of up to about 30 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of up to about 20 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of up to about 10 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody is at a concentration of about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, or about 15 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody that selectively binds to the ECD of nectin-4, such as YMW-1-58, is at a concentration of about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, or about 15 μg / mL. In some embodiments, the rabbit monoclonal α-nectin-4 primary antibody that selectively binds to the ECD of nectin-4, such as YMW-1-58, is at a concentration of about 10 μg / mL.

[0127] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring staining intensity in tumor tissue sections of the patients using a Nectin-4 mIF assay and selecting patients who stain positive in the Nectin-4 mIF assay. In some embodiments, the Nectin-4 mIF assay is as described in Example 3 herein.

[0128] As used herein, the term "patients with positive staining" refers to patients in which a certain percentage of cells in tumor tissue sections stain positive in a Nectin-4 mIF assay. In some embodiments, a patient with positive staining has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of cells in tumor tissue sections stain positive in a Nectin-4 mIF assay.

[0129] There are various methods for measuring staining intensity in mIF assays. In some embodiments, staining intensity is measured by visual scoring, for example, by manual scoring using conventional light microscopy. In some embodiments, staining intensity is measured by computed tissue analysis (CTA) scoring. Staining intensity levels can be either no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+). In some embodiments, staining intensity is measured on tumor cell membranes in tumor tissue sections. In some embodiments, staining intensity is measured in tumor cell cytoplasm in tumor tissue sections. In some embodiments, staining intensity is measured in both tumor cell membranes and tumor cell cytoplasm in tumor tissue sections.

[0130] In some embodiments, positive staining refers to an H-score of about 15 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 20 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 30 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 40 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 50 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 75 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 100 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 125 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to an H-score of about 150 or greater in tumor tissue sections in an mIF assay.

[0131] The H score is the sum of the product of the percentage of cells times the staining intensity of the cells on the 0-3 scale described above (no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+)): [((0 × (0 cells%)) + ((1 × (1+ cells%)) + ((2 × (2+ cells%)) + ((3 × (3 cells%))]

[0132] H-scores can be generated for different sections of a tumor tissue section, including, for example, tumor cell membranes and cytoplasm. In some embodiments, the H-score refers to the tumor cell membrane H-score, which is the sum of the products of the percentage of cells times their membrane staining intensity on a scale of 0 to 3, as described above. In some embodiments, the H-score refers to the tumor cell cytoplasm H-score, which is the sum of the products of the percentage of cells times their cytoplasmic staining intensity on a scale of 0 to 3, as described above.

[0133] In some embodiments, positive staining refers to a tumor cell membrane H-score of about 15 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 20 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 30 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 40 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 50 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 75 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 100 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 125 or greater in tumor tissue sections in an mIF assay. In some embodiments, positive staining refers to a tumor cell membrane H-score of about 150 or greater in tumor tissue sections in an mIF assay.

[0134] In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 15 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 20 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 30 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 40 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 50 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 75 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 100 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to an H-score of tumor cell cytoplasm of about 125 or greater in a tumor tissue section in an mIF assay. In some embodiments, positive staining refers to a tumor cell cytoplasm H-score of about 150 or greater in tumor tissue sections in an mIF assay.

[0135] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring staining intensity in tumor tissue sections of the patients using a Nectin-4 mIF assay, and selecting patients with an H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater.

[0136] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring staining intensity in tumor tissue sections of the patients using a Nectin-4 mIF assay, and selecting patients with a tumor cell membrane H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater.

[0137] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in tumor tissue, comprising measuring staining intensity in tumor tissue sections of the patients using a Nectin-4 mIF assay, and selecting patients with a tumor cell cytoplasm H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater.

[0138] In some embodiments, the present invention provides a method of treating cancer in a patient with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein and / or RNA expression levels are as described herein.

[0139] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein and / or RNA expression levels are as described herein.

[0140] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring Nectin-4 protein and / or RNA expression levels in tumor tissue sections of the patient, selecting a patient with elevated Nectin-4 protein and / or RNA expression levels in the tumor tissue, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein and / or RNA expression levels are as described herein.

[0141] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is ovarian cancer.

[0142] In some embodiments, the present invention provides a method of treating cancer in a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the Nectin-4 mIF assay is as described herein.

[0143] In some embodiments, the present invention provides a method for treating cancer in a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0144] In some embodiments, the present invention provides a method for treating cancer in a patient having a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0145] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising selecting a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0146] In some embodiments, the present invention provides a method for treating cancer in a patient, comprising selecting a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, and administering to the patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0147] In some embodiments, the present invention provides a method for treating cancer in a patient, comprising selecting a patient having a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, and administering to the patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0148] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 mIF assay, selecting patients with an H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0149] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 mIF assay, selecting patients with a tumor cell membrane H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0150] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in a tumor tissue section of the patient using a Nectin-4 mIF assay, selecting patients with a tumor cell cytoplasm H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more, and administering a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to the patient.

[0151] In some embodiments, the bicyclic TICA specific for Nectin-4 is BT7480, as described herein, or a pharmaceutically acceptable salt thereof.

[0152] In some embodiments, the present invention provides a method for treating cancer in a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, comprising administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0153] In some embodiments, the present invention provides a method for treating cancer in a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, comprising administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0154] In some embodiments, the present invention provides a method for treating cancer in a patient, comprising selecting a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, and administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to the patient.

[0155] In some embodiments, the present invention provides methods of treating cancer in a patient, comprising selecting a patient having a tumor cell membrane H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater in a tumor tissue section in an mIF assay, and administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0156] In some embodiments, the present invention provides methods for treating cancer in a patient, comprising measuring Nectin-4 protein and / or RNA expression levels in tumor tissues of the patient, selecting a patient with elevated Nectin-4 protein and / or RNA expression levels in tumor tissues, and administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

[0157] In some embodiments, the present invention provides a method of treating cancer in a patient, comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 mIF assay, selecting patients with a tumor cell membrane H-score of about 15 or greater, about 20 or greater, about 30 or greater, about 40 or greater, about 50 or greater, about 75 or greater, about 100 or greater, about 125 or greater, or about 150 or greater, and administering BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to the patient.

[0158] The Nectin-4 specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be administered to a patient in various dosage ranges.

[0159] In some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dose of about 1 mg / kg or less. In some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dose of about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, or about 0.1 mg / kg.

[0160] In some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dose of about 100 mg / m or less. In some embodiments, the methods of the present invention comprise administering to a patient a Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dose of about 90 mg / m, about 80 mg / m, about 70 mg / m, about 60 mg / m, about 50 mg / m, about 40 mg / m, about 30 mg / m, about 25 mg / m, about 22.5 mg / m, about 20 mg / m, about 17.5 mg / m, about 15 mg / m, about 12.5 mg / m, about 10 mg / m, about 7.5 mg / m, about 5 mg / m, about 2.5 mg / m, or about 1 mg / m. In some embodiments, the methods of the present invention comprise administering to a patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dose of about 2 mg / m to about 25 mg / m.

[0161] The Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof can be administered to a patient at various dosing frequencies. In some embodiments, the method of the present invention comprises administering the Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a patient at a dosing frequency of once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In some embodiments, the method of the present invention comprises administering the Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a patient at a dosing frequency of twice weekly, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[0162] 4. Formulation and Administration In some embodiments, the methods described herein comprise administering a pharmaceutical composition comprising a Nectin-4-specific bicyclic toxin conjugate described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, the Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, is formulated for IV administration to a patient.

[0163] In some embodiments, the methods described herein comprise administering a pharmaceutical composition comprising a Nectin-4-specific bicyclic TICA described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, the Nectin-4-specific bicyclic TICA, or a pharmaceutically acceptable salt thereof, is formulated for IV administration to a patient.

[0164] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, and protamine sulfate, water, partial glyceride mixtures of salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0165] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media.

[0166] For this purpose, any mild, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for preparing injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0167] The pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dry cornstarch.When aqueous suspensions are required for oral use, active ingredients are combined with emulsifiers and suspending agents.If desired, certain sweeteners, flavorings or coloring agents can also be added.

[0168] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0169] The pharmaceutically acceptable compositions of this invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0170] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical-transdermal patches may also be used.

[0171] For topical application, the provided pharmaceutically acceptable composition can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water.Alternatively, the provided pharmaceutically acceptable composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0172] Pharmaceutically acceptable compositions provided for ophthalmic use can be formulated as micronized suspensions in isotonic, pH-adjusted sterile saline, or preferably as solutions in isotonic, pH-adjusted sterile saline, either with or without preservatives such as benzylalkonium chloride. Alternatively, for ophthalmic use, pharmaceutically acceptable compositions can be formulated into an ointment such as petrolatum.

[0173] The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0174] Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0175] The amount of the compounds of the present invention that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host being treated, the particular mode of administration, etc. Preferably, the compositions provided should be formulated so that a patient receiving these compositions can be administered a dosage of between 0.01 and 1 mg / kg body weight / day.

[0176] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compounds employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, as well as the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the invention in the composition will also depend on the particular compound in the composition.

[0177] 5.Use In some embodiments, the present invention provides methods of treating cancer in a patient, comprising selecting a patient with elevated Nectin-4 protein levels in tumor tissue, e.g., using the methods described herein, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the treatment method further comprises measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), e.g., using whole exome sequencing as described herein.

[0178] In some embodiments, the present invention provides a method for treating cancer in a patient with elevated Nectin-4 protein levels in tumor tissue, comprising administering to the patient a Nectin-4-specific bicyclic toxin conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the treatment method further comprises measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), for example, using whole exome sequencing as described herein.

[0179] In some embodiments, the present invention provides methods of treating cancer in a patient, comprising selecting a patient with elevated Nectin-4 protein levels in tumor tissue, e.g., using the methods described herein, and administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the treatment method further comprises measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), e.g., using whole exome sequencing as described herein.

[0180] In some embodiments, the present invention provides a method for treating cancer in a patient with elevated Nectin-4 protein levels in tumor tissue, comprising administering to the patient a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the treatment method further comprises measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), for example, using whole exome sequencing as described herein.

[0181] cancer Cancers or proliferative disorders or tumors that may be treated using the methods and uses described herein include, but are not limited to, hematological cancers, lymphoma, myeloma, leukemia, neurological cancer, skin cancer, breast cancer, prostate cancer, colon cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, renal cancer, and vascular cancer.

[0182] Cancers treated using the methods described herein can be selected from colorectal cancer, including advanced or progressive microsatellite stable (MSS) metastatic colorectal cancer, including microsatellite stable (MSS) CRC; non-small cell lung cancer (NSCLC), including advanced and / or metastatic NSCLC; ovarian cancer; breast cancer, such as inflammatory breast cancer; endometrial cancer; cervical cancer; head and neck cancer; gastric cancer; gastroesophageal junction cancer; and bladder cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is metastatic colorectal cancer. In some embodiments, the colorectal cancer is microsatellite stable (MSS) metastatic colorectal cancer. In some embodiments, the cancer is advanced or progressive microsatellite stable (MSS) CRC. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is advanced and / or metastatic NSCLC. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is inflammatory breast cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is gastroesophageal junction cancer. In some embodiments, the cancer is bladder cancer.

[0183] In some embodiments, cancers include, but are not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangio ... These include endothelial cell carcinoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0184] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0185] In some embodiments, the cancer is acoustic neuroma, astrocytoma (e.g., Grade I—pilocytic astrocytoma, Grade II—low-grade astrocytoma, Grade III—anaplastic astrocytoma, or Grade IV—glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brain stem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET), or schwannoma. In some embodiments, the cancer is a type more commonly found in children than adults, such as brain stem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or pediatric patient.

[0186] In another embodiment, the cancer includes, but is not limited to, mesothelioma, hepatobilliary (liver duct and bile duct), bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal tract (stomach, large intestine, and duodenum), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, cancer, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, non-Hodgkin's lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0187] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine serous adenocarcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepato-bileduct carcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; thyroid anaplastic carcinoma; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain tumor; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0188] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube carcinoma, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatobiliary carcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, thyroid anaplastic carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0189] In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors generally comprise an abnormal mass of tissue that typically does not contain cysts or liquid areas. In some embodiments, the cancer is renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal carcinoma or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial carcinoma, ovarian carcinoma, or fallopian tube carcinoma; papillary serous cystadenocarcinoma or uterine serous adenocarcinoma (UP). SC); prostate cancer; testicular cancer; gallbladder cancer; hepato-bile duct cancer; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; thyroid anaplastic carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain tumor; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0190] In some embodiments, the cancer is selected from renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal carcinoma, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian carcinoma, fallopian tube carcinoma, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatobiliary carcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, thyroid anaplastic carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, brain tumor, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0191] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube carcinoma, ovarian carcinoma, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatobiliary carcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, thyroid anaplastic carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0192] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian carcinoma. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is fallopian tube carcinoma. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine serous adenocarcinoma (UPSC). In some embodiments, the cancer is hepatocholangiocarcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is thyroid anaplastic carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenstrom's macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0193] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, atypical teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, astrocytoma, brain and spinal cord tumor, brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, carcinoma of unknown primary, central nervous system cancer, cervical cancer, Childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, primary fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), Germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular carcinoma, histiocytosis, Langerhans cell carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, AIDS-related lymphoma, macroglobulinemia, male breast cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, malignant mesothelioma, metastatic squamous cell neck cancer of unknown primary site, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloma, multiple myeloma, chronic myeloproliferative disorder, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancerCancer), lip cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus cancer, nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, clear cell renal cell carcinoma, renal pelvis cancer, ureter cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, small intestine cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer of unknown primary site, head and neck squamous cell carcinoma (HNSCC), gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, triple-negative breast cancer (TNBC), gestational trophoblastic tumor, rare childhood cancer of unknown primary site, urethral cancer, uterine cancer, uterine sarcoma, Waldenstrom's macroglobulinemia, or Wilms' tumor.

[0194] In certain embodiments, the cancer is selected from bladder cancer, breast cancer (including TNBC), cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, glioblastoma, head and neck cancer, leukemia (acute and chronic), low-grade glioma, lung cancer (including adenocarcinoma, non-small cell lung carcinoma, squamous cell carcinoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), melanoma, multiple myeloma (MM), ovarian cancer, pancreatic cancer, prostate cancer, renal cancer (including renal clear cell carcinoma and papillary cell renal carcinoma), and gastric cancer.

[0195] In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, colorectal cancer, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), pancreatic cancer, liver cancer, hepatocellular carcinoma, neuroblastoma, other solid tumors or other hematological cancers.

[0196] In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, colon cancer, multiple myeloma, or AML.

[0197] The invention further features methods and compositions for the diagnosis, prognosis, and treatment of virus-associated cancers, including human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16-positive incurable solid tumors, and adult T-cell leukemia, a highly aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by clonal integration of HTLV-I into leukemic cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); and virus-associated tumors in gastric, nasopharyngeal, cervical, vaginal, vulvar, squamous cell carcinoma of the head and neck, and Merkel cell carcinoma. (See https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; see also https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; see also https: / / clinicaltrials.gov / ct2 / show / NCT02426892)

[0198] In some embodiments, the cancer or tumor comprises any of the cancers described herein. In some embodiments, the cancer comprises melanoma. In some embodiments, the cancer comprises breast cancer. In some embodiments, the cancer comprises lung cancer. In some embodiments, the cancer comprises small cell lung cancer (SCLC). In some embodiments, the cancer comprises non-small cell lung cancer (NSCLC).

[0199] In some embodiments, the methods or uses described herein inhibit, reduce, or prevent the growth or spread of cancer or tumor. In some embodiments, the methods or uses described herein inhibit, reduce, or prevent further growth of cancer or tumor. In some embodiments, the methods or uses described herein reduce the size (e.g., volume or mass) of the cancer or tumor by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 99% compared to the size of the cancer or tumor before treatment. In some embodiments, the methods or uses described herein reduce the amount of cancer or tumor in a patient by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 99% compared to the amount of cancer or tumor before treatment.

[0200] The compounds and compositions according to the methods of the present invention can be administered in any amount and using any route of administration effective for treating cancer or tumors or reducing the severity of cancer or tumors. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the disease or condition, the specific drug, its mode of administration, and the like. The compounds and compositions according to the methods of the present invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to a physically discrete unit of drug appropriate for the patient being treated. However, it will be understood that the total daily amount of compounds and compositions to be used will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for a particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field. The term "patient" or "subject", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0201] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powder, ointment, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the disease or disorder being treated. In certain embodiments, the compounds of the present invention can be administered orally or parenterally, one or more times daily, at dosage levels of about 0.01 mg / kg to about 50 mg / kg, preferably about 1 mg / kg to about 25 mg / kg of body weight per day of the subject, to achieve the desired therapeutic effect.

[0202] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0203] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any mild, fixed oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectables.

[0204] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0205] To prolong the effects of the compounds described herein, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends on its dissolution rate, which may in turn depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form can be achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The release rate of the compound can be controlled depending on the compound-to-polymer ratio and the nature of the particular polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0206] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or a suppository wax, which is solid at ambient temperature but liquid at body temperature and thus melts in the rectum or vaginal cavity to release the active compound.

[0207] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicas, and sodium carbonate; e) solution retardants such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0208] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0209] The active compound may also be in microencapsulated form with one or more of the excipients described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. As is customary, such dosage forms may also contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0210] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers, as needed. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. Furthermore, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0211] Co-administration with one or more other therapeutic agent(s) Depending on the particular condition, or disease, being treated, additional therapeutic agents, which are normally administered to treat that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated."

[0212] In some embodiments, the present invention provides methods of treating a disclosed disease or condition, comprising administering to a patient in need thereof an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and simultaneously or sequentially co-administering an effective amount of one or more additional therapeutic agents, such as those described herein. In some embodiments, the method comprises co-administering one additional therapeutic agent to the patient. In some embodiments, the method comprises co-administering two additional therapeutic agents to the patient. In some embodiments, the combination of the disclosed compound and the additional therapeutic agent or agents acts synergistically.

[0213] The compounds of the present invention can also be used in combination with known therapeutic processes, such as the administration of hormones or radiation. In certain embodiments, provided compounds are used as radiosensitizers, particularly for the treatment of tumors that exhibit poor sensitivity to radiation therapy.

[0214] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds, in a possible combination therapy in the form of a fixed combination, or the compounds of the present invention and one or more other therapeutic compounds can be administered at different times, or independently, or in a fixed combination with one or more other therapeutic compounds.The compounds of the present invention can also or additionally be administered in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof, particularly for tumor treatment.As mentioned above, long-term therapy is also possible, as well as adjuvant therapy in conjunction with other treatment strategies.Other possible treatments are, for example, therapy to maintain the patient's condition after tumor regression in at-risk patients, or chemopreventive therapy.

[0215] As part of a multiple dose regimen, one or more other therapeutic agents(s) can be administered separately from the compound or composition of the present invention. Alternatively, one or more other therapeutic agents can be part of a single dosage form, mixed together with the compound of the present invention in a single composition. When administered as a multiple dose regimen, one or more other therapeutic agents(s) and the compound or composition of the present invention can be administered simultaneously, sequentially, or within a period of each other, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, 21, 22, 23, or 24 hours of each other. In some embodiments, one or more other therapeutic agents(s) and the compound or composition of the present invention are administered as a multiple dose regimen, spaced apart by more than 24 hours.

[0216] As used herein, the terms "combination," "in combination," and related terms refer to simultaneous or sequential administration of therapeutic agents according to the present invention. For example, a compound of the present invention can be administered with one or more other therapeutic agents(s) simultaneously or sequentially in separate unit dosage forms, or together in a single unit dosage form. Thus, the present invention provides a single unit dosage form comprising a compound of the present invention, one or more other therapeutic agents(s), and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0217] The amount of a compound of the present invention and one or more other therapeutic agent(s) (in compositions containing additional therapeutic agents as described above) that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated so that a dosage of between 0.01-100 mg / kg body weight / day of a compound of the present invention can be administered.

[0218] In compositions containing one or more other therapeutic agents, the other therapeutic agents and the compounds of the present invention may act synergistically. Thus, the amount of one or more other therapeutic agents in such compositions may be less than the amount required in a monotherapy utilizing only that therapeutic agent. In such compositions, the one or more other therapeutic agents may be administered at a dose of between 0.01 and 1,000 μg / kg body weight / day.

[0219] The amount of one or more other therapeutic agent(s) present in the compositions of the invention can be equal to or less than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of one or more other therapeutic agent(s) in the compositions of the present disclosure ranges from about 50% to about 100% of the amount normally present in a composition comprising that therapeutic agent as the only therapeutic active agent. In some embodiments, the one or more other therapeutic agent(s) is administered at a dose that is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount normally administered for that agent. As used herein, the phrase "normally administered" refers to the amount of an FDA-approved therapeutic agent approved for administration according to the FDA package insert.

[0220] The compounds of the present invention or pharmaceutical compositions thereof can also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, artificial blood vessels, stents, and catheters. For example, vascular stents are used to overcome restenosis (re-narrowing after injury to the blood vessel wall). However, patients using stents or other implantable devices are at risk of thrombus formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. An implantable device coated with the compounds of the present invention is another embodiment of the present invention.

[0221] Exemplary Other Therapeutic Agents In some embodiments, the one or more other therapeutic agents is a poly ADP-ribose polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib (LYNPARZA®, AstraZeneca); rucaparib (RUBRACA®, Clovis Oncology); niraparib (ZEJULA®, Tesaro); talazoparib (MDV3800 / BMN673 / LT00673, Medivation / Pfizer / Biomarin); veliparib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

[0222] In some embodiments, the one or more other therapeutic agents is a histone deacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is selected from vorinostat (ZOLINZA®, Merck); romidepsin (ISTODAX®, Celgene); panobinostat (FARYDAK®, Novartis); belinostat (BELEODAQ®, Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide (EPIDAZA®, HBI-8000, Chipscreen Biosciences, China).

[0223] In some embodiments, the one or more other therapeutic agents is a CDK inhibitor, such as a CDK4 / CDK6 inhibitor. In some embodiments, the CDK4 / 6 inhibitor is selected from palbociclib (IBRANCE®, Pfizer); ribociclib (KISQALI®, Novartis); abemaciclib (Ly2835219, Eli Lilly); and trilaciclib (G1T28, G1 Therapeutics).

[0224] In some embodiments, the one or more other therapeutic agents is a phosphatidylinositol 3-kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is selected from idelalisib (ZYDELIG®, Gilead), alpelisib (BYL719, Novartis), taselisib (GDC-0032, Genentech / Roche); pictilisib (GDC-0941, Genentech / Roche); copanlisib (BAY806946, Bayer); duvelisib (formerly IPI-145, Infinity Pharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202 (formerly RP5230, TG Therapeutics).

[0225] In some embodiments, the one or more other therapeutic agents are platinum-based therapeutic agents, also called platins. Platins cause cross-linking of DNA, inhibiting DNA repair and / or DNA synthesis, primarily in rapidly reproducing cells such as cancer cells. In some embodiments, the platinum-based therapeutic agent is selected from cisplatin (PLATINOL®, Bristol-Myers Squibb); carboplatin (PARAPLATIN®, Bristol-Myers Squibb; also Teva; Pfizer); oxaliplatin (ELOXITIN®, Sanofi-Aventis); nedaplatin (AQUPLA®, Shionogi), picoplatin (Poniard Pharmaceuticals); and satraplatin (JM-216, Agennix).

[0226] In some embodiments, the one or more other therapeutic agents are taxane compounds that cause disruption of microtubules, which are essential for cell division. In some embodiments, the taxane compounds are selected from paclitaxel (TAXOL®, Bristol-Myers Squibb), docetaxel (TAXOTERE®, Sanofi-Aventis, DOCEFREZ®, Sun Pharmaceutical), albumin-bound paclitaxel (ABRAXANE®; Abraxis / Celgene), cabazitaxel (JEVTANA®, Sanofi-Aventis), and SID530 (SK Chemicals, Co.) (NCT00931008).

[0227] In some embodiments, the one or more other therapeutic agents are nucleoside inhibitors or therapeutic agents that interfere with normal DNA synthesis, protein synthesis, cell replication, or otherwise inhibit rapidly proliferating cells.

[0228] In some embodiments, the nucleoside inhibitor is trabectedin (a guanidine alkylating agent, YONDELIS®, Janssen Oncology), mechlorethamine (an alkylating agent, VALCHLOR®, Aktelion Pharmaceuticals), vincristine (ONCOVIN®, Eli Lilly; VINCASAR®, Teva Pharmaceuticals; MARQIBO®, Talon Therapeutics); temozolomide (a prodrug of the alkylating agent 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC) TEMODAR®, Merck); cytarabine injection (ara-C, an antimetabolite cytidine analog, Pfizer); lomustine (an alkylating agent, CEENU®, Bristol-Myers Squibb; GLEOSTINE®, NextSource) Biotechnology); azacitidine (a pyrimidine nucleoside analog of cytidine, VIDAZA®, Celgene); omacetaxine mepeccinate (cephalotaxine ester) (a protein synthesis inhibitor, SYNRIBO)®; Teva Pharmaceuticals); asparaginase Erwinia chrysanthemi (an asparagine-removing enzyme, ELSPAR®, Lundbeck; ERWINAZE®, EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based antimitotic agent, HALAVEN®, Eisai); cabazitaxel (microtubule inhibitor, tubulin-based antimitotic agent, JEVTANA®, Sanofi-Aventis); capsetrin (thymidylate synthase inhibitor, XELODA®, Genentech); bendamustine (bifunctional mechlorethamine derivative, thought to form interstrand DNA crosslinks, TREANDA®, Cephalon / Teva); ixabepilone (semi-synthetic analog of epothilone B, microtubule inhibitor, tubulin-based antimitotic agent, IXEMPRA®, Bristol-Myers Squibb);Selected from nelarabine (prodrug of a deoxyguanosine analog, a nucleoside metabolic inhibitor, ARRANON®, Novartis); chlorafavine (prodrug of a ribonucleotide reductase inhibitor, a competitive inhibitor of deoxycytidine, CLOLAR®, Sanofi-Aventis); and trifluridine and tipiracil (thymidine-based nucleoside analog and thymidine phosphorylase inhibitor, LONSURF®, Taiho Oncology);

[0229] In some embodiments, the one or more other therapeutic agents are kinase inhibitors or VEGF-R antagonists. Approved VEGF inhibitors and kinase inhibitors useful in the present invention include bevacizumab (AVASTIN®, Genentech / Roche), an anti-VEGF monoclonal antibody; ramucirumab (CYRAMZA®, Eli Lilly), an anti-VEGFR-2 antibody, and aflibercept, also known as VEGFTrap (ZALTRAP®; Regeneron / Sanofi), VEGFR inhibitors such as regorafenib (STIVARGA®, Bayer; vandetanib (CAPRELSA®, AstraZeneca); axitinib (INLYTA®, Pfizer); and lenvatinib (LENVIMA®, Eisai); Raf inhibitors such as sorafenib (NEXAVAR®, Bayer AG and Onyx; dabrafenib (TAFINLAR®, Novartis); and vemurafenib (ZELBORAF®, Genentech / Roche); MEK inhibitors, such as cobimetanib (COTELLIC®, Exelexis / Genentech / Roche); trametinib (MEKINIST®, Novartis); Bcr-Abl tyrosine kinase inhibitors, such as imatinib (GLEEVEC®, Novartis); nilotinib (TASIGNA®, Novartis); dasatinib (SPRYCEL®, BristolMyersSquibb); bosutinib (BOSULIF®, Pfizer); and ponatinib (INCLUSIG®, Ariadna) Pharmaceuticals; Her2 and EGFR inhibitors, e.g., gefitinib (IRESSA®, AstraZeneca); erlotinib (TARCEEVA®, Genentech / Roche / Astellas); lapatinib (TYKERB®, Novartis); afatinib (GILOTRIF®, Boehringer Ingelheim);Examples of inhibitors include osimertinib (TAGRISSO®, AstraZeneca, which targets activated EGFR); and brigatinib (ALUNBRIG®, Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozantinib (COMETRIQ®, Exelexis); and multikinase inhibitors, such as sunitinib (SUTENT®, Pfizer); pazopanib (VOTRIENT®, Novartis); ALK inhibitors, such as crizotinib (XALKORI®, Pfizer); ceritinib (ZYKADIA®, Novartis); and alectinib (ALECENZa®, Genentech / Roche); Bruton's tyrosine kinase inhibitors, such as ibrutinib (IMBRUVICA®, Pharmacyclics / Janssen); and Flt3 receptor inhibitors, such as midostaurin (RYDAPT®, Novartis). ;

[0230] Other kinase inhibitors and VEGF-R antagonists under development that can be used in the present invention include tivozanib (Aveo Pharmaceuticals); vatalanib (Bayer / Novartis); lucitanib (Clovis Oncology); dovitinib (TKI258, Novartis); chiauanib (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories); neratinib (HKI-272, Puma Biotechnology); radotinib (SUPECT®, IY5511, Il-Yang Pharmaceuticals, S.Korea); ruxolitinib (JAKAFI®, Incyte Corporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo) and motesanib (Amgen / Takeda).

[0231] In some embodiments, the one or more other therapeutic agents are mTOR inhibitors that inhibit cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitors are everolimus (AFINITOR®, Novartis); temsirolimus (TORISEL®, Pfizer); and sirolimus (RAPAMUNE®, Pfizer).

[0232] In some embodiments, the one or more other therapeutic agents is a proteasome inhibitor. Approved proteasome inhibitors useful in the present invention include bortezomib (VELCADE®, Takeda); carfilzomib (KYPROLIS®, Amgen); and ixazomib (NINLARO®, Takeda).

[0233] In some embodiments, the one or more other therapeutic agents are growth factor antagonists, such as antagonists of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) or its receptor (EGFR). Approved PDGF antagonists that can be used in the present invention include olatumab (LARTRUVO®; Eli Lilly). Approved EGFR antagonists that can be used in the present invention include cetuximab (ERBITUX®, Eli Lilly); necitumumab (PORTRAZZA®, Eli Lilly), panitumumab (VECTIBIX®, Amgen); and osimertinib (targeting activated EGFR, TAGRISSO®, AstraZeneca).

[0234] In some embodiments, the one or more other therapeutic agents is an aromatase inhibitor. In some embodiments, the aromatase inhibitor is selected from exemestane (AROMASIN®, Pfizer); anastazole (ARIMIDEX®, AstraZeneca), and letrozole (FEMARA®, Novartis).

[0235] In some embodiments, the one or more other therapeutic agents are hedgehog pathway antagonists. Approved hedgehog pathway inhibitors that can be used in the present invention include sonidegib (ODOMZO®, Sun Pharmaceuticals); and vismodegib (ERIVEDGE®, Genentech), both for the treatment of basal cell carcinoma.

[0236] In some embodiments, the one or more other therapeutic agents is a folate inhibitor. Approved folate inhibitors useful in the present invention include pemetrexed (ALIMTA®, Eli Lilly).

[0237] In some embodiments, the one or more other therapeutic agents are CC chemokine receptor 4 (CCR4) inhibitors. Investigational CCR4 inhibitors that may be useful in the present invention include mogamulizumab (POTELIGEO®, Kyowa Hakko Kirin, Japan).

[0238] In some embodiments, the one or more other therapeutic agents are isocitrate dehydrogenase (IDH) inhibitors. Investigational IDH inhibitors that can be used in the present invention include AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); IDH305 (Novartis, NCT02987010).

[0239] In some embodiments, the one or more other therapeutic agents are arginase inhibitors. Investigational arginase inhibitors that can be used in the present invention include AEB1102 (pegylated recombinant arginase, Aeglea Biotherapeutics), which is being studied in Phase 1 clinical trials for acute myeloid leukemia and myelodysplastic syndromes (NCT02732184) and solid tumors (NCT02561234), and CB-1158 (Calithera Biosciences).

[0240] In some embodiments, the one or more other therapeutic agents are glutaminase inhibitors. Investigational glutaminase inhibitors that can be used in the present invention include CB-839 (Calithera Biosciences).

[0241] In some embodiments, the one or more other therapeutic agents are antibodies that bind to tumor antigens, i.e., proteins expressed on the cell surface of tumor cells. Approved antibodies that bind to tumor antigens that can be used in the present invention include rituximab (RITUXAN®, Genentech / BiogenIdec); ofatumumab (anti-CD20, ARZERRA®, GlaxoSmithKline); obinutuzumab (anti-CD20, Gazyva®, Genentech), ibritumomab (anti-CD20 and yttrium-90, ZEVALIN®, Spectrum Pharmaceuticals); daratumumab (anti-CD38, DARZALEX®, Janssen Biotech), dinutuximab (anti-glycolipid GD2, UNITUXIN®, United Therapeutics); trastuzumab (anti-HER2, HERCEPTIN®, Genentech); ado-trastuzumab emtansine (anti-HER2, fused to emtansine, KADCYLA®, Genentech); and pertuzumab (anti-HER2, PERJETA®, Genentech); and brentuximab vedotin (anti-CD30 drug conjugate, ADCETRIS®, Seattle Genetics).

[0242] In some embodiments, the one or more other therapeutic agents are topoisomerase inhibitors. Approved topoisomerase inhibitors useful in the present invention include irinotecan (ONIVYDE®, Merrimack Pharmaceuticals); topotecan (HYCAMTIN®, GlaxoSmithKline). Investigational topoisomerase inhibitors that can be used in the present invention include pixantrone (PIXUVRI®, CTI Biopharma).

[0243] In some embodiments, the one or more other therapeutic agents are inhibitors of anti-apoptotic proteins, such as BCL-2. Approved anti-apoptotic agents that can be used in the present invention include venetoclax (VENCLEXTA®, AbbVie / Genentech); and blinatumomab (BLINCYTO®, Amgen). Other therapeutic agents targeting apoptotic proteins undergoing clinical trials that can be used in the present invention include navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

[0244] In some embodiments, the one or more other therapeutic agents are androgen receptor inhibitors. Approved androgen receptor inhibitors useful in the present invention include enzalutamide (XTANDI®, Astellas / Medivation); approved inhibitors of androgen synthesis include abiraterone (ZYTIGA®, Centocor / Ortho); approved antagonists of the gonadotropin-releasing hormone (GnRH) receptor include (degalarix, FIRMAGON®, Ferring Pharmaceuticals).

[0245] In some embodiments, the one or more other therapeutic agents are selective estrogen receptor modulators (SERMs) that interfere with the synthesis or activity of estrogen. Approved SERMs useful in the present invention include raloxifene (EVISTA®, Eli Lilly).

[0246] In some embodiments, the one or more other therapeutic agents are inhibitors of bone resorption. An approved therapeutic agent that inhibits bone resorption is denosumab (XGEVA®, Amgen), an antibody that binds to RANKL and prevents it from binding to its receptor RANK, which is found on the surface of osteoclasts, their precursor cells, and osteoclast-like giant cells and mediates bone lesions of solid tumors with bone metastasis. Other approved therapeutic agents that inhibit bone resorption include bisphosphonates, such as zoledronic acid (ZOMETA®, Novartis).

[0247] In some embodiments, the one or more other therapeutic agents are inhibitors of the interaction between MDMX and MDM2, two major p53 suppressor proteins. Investigational inhibitors of p53 suppressor proteins that can be used in the present invention include ALRN-6924 (Aileron), a stapled peptide that binds equivalently to MDMX and MDM2 and disrupts the interaction of MDMX and MDM2 with p53. ALRN-6924 is currently being evaluated in clinical trials (NCT02909972; NCT02264613) for the treatment of AML, i.e., advanced myelodysplastic syndrome (MDS), and peripheral T-cell lymphoma (PTCL).

[0248] In some embodiments, the one or more other therapeutic agents are inhibitors of transforming growth factor beta (TGF beta or TGFβ). Investigational inhibitors of TGF-beta proteins that can be used in the present invention include NIS793 (Novartis), an anti-TGF-beta antibody being clinically tested for the treatment of various cancers, including breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer (NCT02947165). In some embodiments, the TGF-beta protein inhibitor is fresolimumab (GC1008; Sanofi-Genzyme), which is being tested for melanoma (NCT00923169); renal cell carcinoma (NCT00356460); and non-small cell lung cancer (NCT02581787). Furthermore, in some embodiments, the additional therapeutic agent is a TGF-beta trap, as described in Connolly et al. (2012) Int'l J. Biological Sciences 8:964-978. One therapeutic compound currently in clinical trials for the treatment of solid tumors is M7824 (Merck KgaA - formerly MSB0011459X), a bispecific anti-PD-L1 / TGFβ trap compound (NCT02699515); and (NCT02517398). M7824 consists of a fully human IgG1 antibody against PD-L1 fused to the extracellular domain of human TGF-beta receptor II, which functions as a TGFβ "trap."

[0249] In some embodiments, the one or more other therapeutic agents are selected from glenbatumumab vedotin-monomethylauristatin E (MMAE) (Celldex), an anti-glycoprotein NMB (gpNMB) antibody linked to the cytotoxic MMAE (CR011). gpNMB is a protein overexpressed in multiple tumor types that is associated with the metastatic potential of cancer cells.

[0250] In some embodiments, the one or more other therapeutic agents are antiproliferative compounds. Such antiproliferative compounds include aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antitumor antimetabolites; platinum compounds; compounds that target / reduce protein or lipid kinase activity and additional antiangiogenic compounds; compounds that target, reduce, or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; antiandrogens; methionine amines. peptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematological malignancies; compounds that target, decrease, or inhibit the activity of Flt-3; Hsp90 inhibitors, such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, Conforma temozolomide (TEMODAL®); kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine / chlorpromazine from CombinatoRx; MEK inhibitors, such as ARRY142886 from Array BioPharma, AZd6244 from AstraZeneca, PD181461 from Pfizer, and leucovorin.

[0251] The term "aromatase inhibitor" as used herein refers to compounds that inhibit estrogen production, such as the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. This term includes, but is not limited to, steroids, particularly atamestane, exemestane, and formestane, and nonsteroids, particularly aminoglutethimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole, and letrozole. Exemestane is sold under the trade name AROMASIN™. Formestane is sold under the trade name LENTARON™. Fadrozole is sold under the trade name AFEMA™. Anastrozole is sold under the trade name ARIMIDEX™. Letrozole is sold under the trade name FEMARA™ or FEMAr™. Aminoglutethimide is sold under the trade name ORIMETEN™. Combinations of the present invention which include a chemotherapeutic agent that is an aromatase inhibitor are particularly useful in the treatment of hormone receptor positive tumors, such as breast tumors.

[0252] The term "antiestrogen" as used herein refers to a compound that antagonizes the effects of estrogen at the estrogen receptor level. This term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen is sold under the trade name NOLVADEX®. Raloxifene hydrochloride is sold under the trade name EVISTA®. Fulvestrant can be administered under the trade name FASLODEX®. The combination of the present invention, which includes a chemotherapeutic agent that is an antiestrogen, is particularly useful for treating estrogen receptor-positive tumors, such as breast tumors.

[0253] As used herein, the term "antiandrogen" refers to any substance capable of inhibiting the biological effects of androgen hormones, including, but not limited to, bicalutamide (CASODEX™). As used herein, the term "gonadorelin agonist" includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin can be administered under the trade name ZOLADEX™.

[0254] The term "topoisomerase I inhibitor" as used herein includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin, and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, for example, in the form as it is marketed, e.g., under the trademark CAMPTOSAR™. Topotecan is sold under the trade name HYCAMPTIN™.

[0255] The term "topoisomerase II inhibitors" as used herein includes, but is not limited to, doxorubicin (including liposomal formulations such as CAELYX™), anthracyclines such as daunorubicin, epirubicin, idarubicin, and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophyllotoxins etoposide and teniposide. Etoposide is sold under the trade name ETOPOPHOS™. Teniposide is commercially available under the trade name VM26-Bristol. Doxorubicin is commercially available under the trade name ACRIBLASTIN™ or ADRIAMYCIN™. Epirubicin is sold under the trade name FARMORUBICIN™. Idarubicin is sold under the trade name ZAVEDOS™. Mitoxantrone is sold under the trade name NOVANTRON™.

[0256] The term "microtubule active agent" refers to microtubule stabilizing compounds, microtubule destabilizing compounds, and microtubule polymerization inhibitors, including taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolide; coticin and epothilones and their derivatives. Paclitaxel is sold under the trade name TAXOL™. Docetaxel is sold under the trade name TAXOTERE™. Vinblastine sulfate is sold under the trade name VINBLASTIN RP™. Vincristine sulfate is sold under the trade name FARMISTIN™.

[0257] The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosoureas (BCNU or Gliadel). Cyclophosphamide is sold under the trade name CYCLOSTIN™. Ifosfamide is sold under the trade name HOLOXAN™.

[0258] The term "histone deacetylase inhibitors" or "HDAC inhibitors" relates to compounds which inhibit histone deacetylase and which possess antiproliferative activity, including, but not limited to, suberoylanilide hydroxamic acid (SAHA).

[0259] The term "antineoplastic antimetabolite" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folate antagonists such as pemetrexed. Capecitabine is sold under the trade name XELODA™. Gemcitabine is sold under the trade name GEMZAR™.

[0260] The term "platinum compounds" as used herein includes, but is not limited to, carboplatin, cisplatin, cisplatinum, and oxaliplatin. Irinotecan can be administered, e.g., in the form as it is marketed, e.g., under the trademark CAMPTOSAR™. Carboplatin can be administered, e.g., in the form as it is marketed, e.g., under the trademark ELOXATIN™.

[0261] The term "compounds that target / increase protein or lipid kinase activity; or protein or lipid phosphatase activity; or further anti-angiogenic compounds" as used herein includes, but is not limited to, protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, for example: a) compounds that target platelet-derived growth factor receptor (PDGFR) and reduce or inhibit its activity, for example compounds that target PDGFR and reduce or inhibit its activity, such as compounds that inhibit PDGF receptors, particularly N-phenyl-2-pyrimidine-amine derivatives such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target fibroblast growth factor receptor (FGFR) and reduce or inhibit its activity; c) compounds that target insulin-like growth factor receptor I (IGF-IR) and reduce or inhibit its activity, for example the kinase activity of IGF-I receptor in particular. or antibodies targeting the IGF-I receptor or the extracellular domain of that growth factor; d) compounds that target the Trk receptor tyrosine kinase family and reduce or inhibit their activity, or ephrin B4 inhibitors; e) compounds that target the AxI receptor tyrosine kinase family and reduce or inhibit their activity; f) compounds that target the Ret receptor tyrosine kinase and reduce or inhibit its activity; g) compounds that target the Kit / SCFR receptor tyrosine kinase and reduce or inhibit its activity, such as imatinib; h) compounds that target the c-Kit receptor tyrosine kinase and reduce or inhibit its activity, for example compounds that target the C-kit receptor tyrosine kinase family, which is part of the PDGFR family, and reduce or inhibit its activity, such as compounds that inhibit the c-Kit receptor, in particular imatinib;i) Compounds that target and reduce or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinase) and mutants, e.g., N-phenyl-2-pyrimidine-amine derivatives such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825), which target and reduce the activity of c-Abl family members and their gene fusion products. j) compounds that target, decrease or inhibit the activity of members of the cyclin-dependent kinase family (CDK), including members of the protein kinase C (PKC) and Raf families of serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families, and / or staurosporine derivatives such as midostaurin; further examples of compounds include , UCN-01, safingol, BAY43-9006, bryostatin 1, perifosine; llmofosine; RO318220 and RO320432; GO6976; lsis3521; LY333531 / LY379196; isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); k) compounds that target protein tyrosine kinase inhibitors, reduce or inhibit their activity. , for example, imatinib mesylate (GLEEVEC™) or tyrphostins, such as tyrphostin A23 / RG-50810; AG99; tyrphostin AG213; tyrphostin AG1748; tyrphostin AG490; tyrphostin B44; tyrphostin B44 (+) enantiomer; tyrphostin AG555; AG494; tyrphostin AG556, AG957, etc., and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;l) compounds that target and reduce or inhibit the activity of protein-tyrosine kinase inhibitors, including NSC680410, adaphostin; l) compounds that target and reduce or inhibit the activity of the epidermal growth factor family of receptor tyrosine kinases (as homodimers or heterodimers (EGFR1, ErbB2, ErbB3, ErbB4) and their variants, e.g., compounds that target and reduce or inhibit the activity of the epidermal growth factor receptor family, in particular compounds, proteins or antibodies that inhibit members of the EGF receptor tyrosine kinase family, such as EGF receptor, ErbB2, ErbB3 and ErbB4, or that bind to EGF or EGF-related ligands, CP358774, ZD1839, ZM105180; trastuzumab (HERCEPTIN™), cetuximab (ERBITUX™), Iressa, Tarceva, OSI-774, Cl-1033 , EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3 and 7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds that target the c-Met receptor and reduce or inhibit its activity, for example compounds that specifically inhibit the kinase activity of the c-Met receptor, or compounds that target c-Met, such as antibodies that target the extracellular domain of c-Met or that bind to HGF. n) compounds that target one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan-JAK) and reduce or inhibit their kinase activity, including, but not limited to, PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, and ruxolitinib;o) compounds that target PI3 kinase (PI3K) and decrease or inhibit its kinase activity, including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib; and q) protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, such as compounds that target, decrease or inhibit the signaling action of the hedgehog protein (Hh) or smoothened receptor (SMO) pathway, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib, and IPI-926 (salidegib);

[0262] The term "PI3K inhibitor" as used herein includes, but is not limited to, compounds that have inhibitory activity against one or more enzymes of the phosphatidylinositol-3-kinase family, such as, but not limited to, PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87. Examples of PI3K inhibitors useful in the present invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib.

[0263] As used herein, the term "Bcl-2 inhibitor" includes, but is not limited to, compounds that have inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including, but not limited to, ABT-199, ABT-731, ABT-737, apogossypol, the pan-Bcl-2 inhibitors of Ascenta, curcumin (and analogs thereof), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and analogs thereof; see WO2008118802), navitoclax (and analogs thereof, see US7390799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and analogs thereof, see WO2004106328), S-001 (Gloria Pharmaceuticals), the TW series of compounds (University of Michigan), and venetoclax. In some embodiments, the Bcl-2 inhibitor is a small molecule therapeutic. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic.

[0264] The term "BTK inhibitor" as used herein includes, but is not limited to, compounds that have inhibitory activity against Bruton's tyrosine kinase (BTK), including, but not limited to, AVL-292 and ibrutinib.

[0265] The term "SYK inhibitor" as used herein includes, but is not limited to, compounds that have inhibitory activity against spleen tyrosine kinase (SYK), including, but not limited to, PRT-062070, R-343, R-333, Excelair, PRT-062607, and fostamatinib.

[0266] Further examples of BTK inhibitor compounds and conditions treatable by such compounds in combination with the compounds of the present invention can be found in WO2008039218 and WO2011090760, which are incorporated herein by reference in their entireties.

[0267] Further examples of SYK inhibitor compounds, and conditions treatable by such compounds in combination with the compounds of the present invention, can be found in WO2003063794, WO2005007623, and WO2006078846, which are incorporated herein by reference in their entireties.

[0268] Further examples of PI3K inhibitor compounds and conditions treatable by such compounds in combination with the compounds of the present invention can be found in WO2004019973, WO2004089925, WO2007016176, US8138347, WO2002088112, WO2007084786, WO2007129161, WO2006122806, WO2005113554, and WO2007044729, the entire contents of which are incorporated herein by reference.

[0269] Further examples of JAK inhibitor compounds and conditions treatable by such compounds in combination with the compounds of the present invention can be found in WO2009114512, WO2008109943, WO2007053452, WO2000142246, and WO2007070514, the entireties of which are incorporated herein by reference.

[0270] Additional anti-angiogenic compounds include compounds that have another mechanism for their activity unrelated to, for example, protein or lipid kinase inhibition, such as thalidomide (THALOMID™) and TNP-470.

[0271] Examples of proteasome inhibitors useful in combination with the compounds of the invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0272] Compounds which target protein or lipid phosphatases, decrease or inhibit their activity are eg inhibitors of phosphatase 1, phosphatase 2A or CDC25, eg okadaic acid or a derivative thereof.

[0273] Compounds that induce cell differentiation processes include, but are not limited to, retinoic acid, α-, γ- or δ-tocopherol, or α-, γ- or δ-tocotrienol.

[0274] The term cyclooxygenase inhibitors as used herein includes Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acids and derivatives, such as celecoxib (CELEBREX™), rofecoxib (VIOXX™), etoricoxib, and valdecoxib, or 5-alkyl-2-arylaminophenylacetic acids, such as 5-methyl-2-(2′-chloro-6′-fluoroanilino)phenylacetic acid and lumiracoxib.

[0275] The term "bisphosphonate" as used herein includes, but is not limited to, etridonic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid. Etridonic acid is sold under the trade name DIDRONEL™. Clodronic acid is sold under the trade name BONEFOS™. Tiludronic acid is sold under the trade name Skelid™. Pamidronic acid is sold under the trade name AREDIA™. Alendronic acid is sold under the trade name FOSAMAX™. Ibandronic acid is sold under the trade name BONDRANAT™. Risedronic acid is sold under the trade name ACTONEL™. Zoledronic acid is sold under the trade name ZOMETA™. The term "mTOR inhibitors" relates to compounds which inhibit the mammalian target of rapamycin (mTOR) and have antiproliferative activity, such as sirolimus (RAPAMUNE®), everolimus (CERTICAN™), CCI-779 and ABT578.

[0276] As used herein, the term "heparanase inhibitor" refers to a compound that targets, decreases, or inhibits acid heparin degradation. This term includes, but is not limited to, PI-88. As used herein, the term "biological response modifier" refers to lymphokines or interferons.

[0277] As used herein, the term "inhibitor of Ras oncogenic isoforms" such as H-Ras, K-Ras, or N-Ras refers to compounds that target, reduce, or inhibit the oncogenic activity of Ras, such as "farnesyltransferase inhibitors" such as L-744832, DK8G557, or R115777 (ZARNESTRA®). As used herein, the term "telomerase inhibitor" refers to compounds that target, reduce, or inhibit telomerase activity. Compounds that target, reduce, or inhibit telomerase activity are particularly compounds that inhibit telomerase receptors, such as telomestatin.

[0278] The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets methionine aminopeptidase and reduces or inhibits its activity.Methionine aminopeptidase targeting compounds that reduces or inhibits its activity include but are not limited to bengamide or its derivatives.

[0279] As used herein, the term "proteasome inhibitor" refers to a compound that targets the proteasome, decreasing or inhibiting its activity. Compounds that target the proteasome, decreasing or inhibiting its activity include, but are not limited to, bortezomib (VELCADE™) and MLN 341.

[0280] The term "matrix metalloproteinase inhibitors" or ("MMP" inhibitors) as used herein includes, but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors, tetracycline derivatives, such as the hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogs marimastat (BB-2516), prinomastat (AG3340), metastat (NSC683551), BMS-279251, BAY12-9566, TAA211, MMI270B, or AAJ996.

[0281] As used herein, the term "compounds used in the treatment of hematological malignancies" includes FMS-like tyrosine kinase inhibitors, which are compounds that target, decrease or inhibit the FMS-like tyrosine kinase receptor (Flt-3R); interferon, 1-β-D-arabinofuransylcytosine (ara-c), and busulfan; and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

[0282] Compounds that target the FLT-3R tyrosine kinase receptor (Flt-3R) and decrease or inhibit its activity are, in particular, compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518.

[0283] The term "HSP90 inhibitors" as used herein includes, but is not limited to, compounds that target, decrease, or inhibit the intrinsic ATPase activity of HSP90; compounds that degrade, target, decrease, or inhibit HSP90 client proteins via the ubiquitin proteosome pathway. Compounds that target, decrease, or inhibit the intrinsic ATPase activity of HSP90 are, in particular, compounds, proteins, or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives, other geldanamycin-related compounds, radicicol, and HDAC inhibitors.

[0284] As used herein, the term "antiproliferative antibody" includes, but is not limited to, trastuzumab (HERCEPTIN™), trastuzumab-DM1, erbitux, bevacizumab (AVASTIN™), rituximab (RITUXAN®), PRO64553 (anti-CD40), and 2C4 antibodies. Antibody refers to intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0285] For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia treatments, particularly in combination with treatments used to treat AML. In particular, the compounds of the present invention can be administered in combination with, for example, farnesyltransferase inhibitors and / or other drugs useful for the treatment of AML, such as daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatinum, and PKC412.

[0286] Other anti-leukemia compounds include, for example, Ara-C, a pyrimidine analog, which is a 2'-alpha-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are the purine analog of hypoxanthine, 6-mercaptopurine (6-MP), and fludarabine phosphate. Compounds that target and reduce or inhibit the activity of histone deacetylase (HDAC) inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), trichostatin A, and compounds disclosed in US Pat. No. 6,552,065, including, but not limited to, N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, particularly the lactate salt. As used herein, somatostatin receptor antagonist refers to compounds that target, treat, or inhibit somatostatin receptors, such as octreotide and SOM230. Tumor cell damaging approaches refer to approaches such as ionizing radiation. The term "ionizing radiation," as referred to above and hereinafter, means ionizing radiation that occurs either as electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but is not limited to, radiation therapy, as known in the art. See Hellman, "Principles of Radiation Therapy," Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4th Edition, Vol. 1, pp. 248-275 (1993).

[0287] Also included are EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressants that modulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitor" refers to a pyrimidine or purine nucleoside analog, including, but not limited to, fludarabine and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C for ALL), and / or pentostatin. Ribonucleotide reductase inhibitors are, in particular, hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0288] Also included are VEGF compounds, proteins or monoclonal antibodies, such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate, ANGIOSTATIN™; ENDOSTATIN™; anthranilic acid amide; ZD4190; Zd6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as VEGF aptamers such as rhuMAb and RHUFab, Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, Angiozyme (RPI 4610) and bevacizumab (AVASTIN™).

[0289] As used herein, photodynamic therapy refers to the use of certain chemicals known as photosensitizing compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as VISUDYNE™ and porfimer sodium.

[0290] As used herein, angiogenic antisteroids refer to compounds that block or inhibit angiogenesis, such as anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocortisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, and dexamethasone.

[0291] Corticosteroid-containing implants refer to compounds such as fluocinolone and dexamethasone.

[0292] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or miscellaneous compounds or compounds with other or unknown mechanisms of action.

[0293] The structures of the active compounds identified by code numbers, generic names or trade names can be taken from the actual edition of the standard abstract "The Merck Index" or from databases such as Patents International (e.g. IMS World Publications).

[0294] Exemplary Immuno-Oncology Agents In some embodiments, the one or more other therapeutic agents are immuno-oncology agents. As used herein, the term "immuno-oncology agent" refers to an agent that is effective in enhancing, stimulating, and / or upregulating the immune response in a subject. In some embodiments, administration of an immuno-oncology agent with a compound of the present invention has a synergistic effect in the treatment of cancer.

[0295] The immuno-oncology agent may be, for example, a small molecule drug, an antibody, or a biological or small molecule. Examples of biological immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is humanized or human.

[0296] In some embodiments, the immuno-oncology agent is (i) an agonist of a stimulatory (including costimulatory) receptor, or (ii) an antagonist of an inhibitory (including costimulatory) signal on T cells, both of which result in amplification of antigen-specific T cell responses.

[0297] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory or costimulatory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3 , EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0298] In some embodiments, the immuno-oncology agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines) or a cytokine that stimulates T cell activation to stimulate an immune response.

[0299] In some embodiments, the combination of a compound of the present invention with an immuno-oncology agent can stimulate a T cell response. In some embodiments, the immuno-oncology agent is (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, or (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0300] In some embodiments, the immuno-oncology agent is an antagonist of an inhibitory receptor on NK cells or an agonist of an activating receptor on NK cells. In some embodiments, the immuno-oncology agent is an antagonist of KIR, such as lirilumab.

[0301] In some embodiments, the immuno-oncology agent is an agent that inhibits or depletes macrophages or monocytes, including, but not limited to, CSF-1R antagonists such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0302] In some embodiments, the immuno-oncology agent is selected from agonists that ligate positive costimulatory receptors, blocking agents (i.e., antagonists) that attenuate signaling through inhibitory receptors, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibiting metabolic enzymes such as IDO, or reversing / preventing T cell anergy or exhaustion), and agents that cause innate immune activation and / or inflammation at the tumor site.

[0303] In some embodiments, the immuno-oncology agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, the antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab.

[0304] In some embodiments, the immuno-oncology agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immuno-oncology agent is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO 2012 / 145493). In some embodiments, the immuno-oncology agent may be pidilizumab (CT-011). In some embodiments, the immuno-oncology agent is a recombinant protein called AMP-224, which is composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1.

[0305] In some embodiments, the immuno-oncology agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, the PD-L1 antibody is atezolizumab (MPDL3280A, RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), avelumab (MSB0010718C, WO2013 / 791740), or cemiplimab (REGN2810).

[0306] In some embodiments, the immuno-oncology agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, the LAG-3 antibody is BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO009 / 44273).

[0307] In some embodiments, the immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonistic CD137 antibody. In some embodiments, the CD137 antibody is uremab or PF-05082566 (WO12 / 32433).

[0308] In some embodiments, the immuno-oncology agent is a GITR agonist. In some embodiments, the GITR agonist is an agonistic GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO006 / 105021, WO009 / 009116), or MK-4166 (WO11 / 028683).

[0309] In some embodiments, the immuno-oncology agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); an enzyme that breaks down kynurenine (Kynase, IKENA Oncology, formerly known as Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0310] In some embodiments, the immuno-oncology agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic anti-human OX40 antibody. In some embodiments, the OX40 agonist antibody is MEDI-6383 or MEDI-6469.

[0311] In some embodiments, the immuno-oncology agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. The OX40L antagonist is RG-7888 (WO06 / 029879).

[0312] In some embodiments, the immuno-oncology agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the immuno-oncology agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0313] In some embodiments, the immuno-oncology agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is valilumab.

[0314] In some embodiments, the immuno-oncology agent is MGA271 (against B7H3) (WO11 / 109400).

[0315] In some embodiments, the immuno-oncology agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab, mafenatox mafenatox, apolizumab, atezolizumab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[0316] In some embodiments, the immuno-oncology agent is an immunostimulatory agent. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can liberate activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in an increasing number of tumor histologies, including multiple tumor types not previously considered susceptible to immunotherapy. See, e.g., Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al. (2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab (OPDIVO®, Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558) has shown the potential to improve overall survival in RCC patients who have experienced disease progression during or after previous antiangiogenic therapy.

[0317] In some embodiments, the immunomodulatory therapeutic specifically induces apoptosis in tumor cells. Approved immunomodulatory therapeutics that can be used in the present invention include pomalidomide (POMALYST®, Celgene); lenalidomide (REVLIMID®, Celgene); ingenol mebutate (PICATO®, LEO Pharma).

[0318] In some embodiments, the immuno-oncology agent is a cancer vaccine, hi some embodiments, the cancer vaccine is selected from sipuleucel-T (PROVENGE®, Dendreon / Valeant Pharmaceuticals), which is approved for the treatment of asymptomatic, or minimally symptomatic, metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (IMLYGIC®, BioVex / Amgen, formerly known as T-VEC), a gene-modified oncolytic virus therapy approved for the treatment of unresectable cutaneous, subcutaneous, and lymph node disease in melanoma.In some embodiments, the immuno-oncology agent is an oncolytic virus therapy for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312), e.g., pexaVec / JX-594 (SillaJen / formerly Jennerex-Biotherapeutics), a thymidine kinase- (TK-)-deficient vaccinia engineered to express GM-CSF. pelareorep (REOLYSIN®, Oncolytics-Biotech), a novel anti-RAS inhibitor, has been shown to inhibit RAS activation in multiple cancers, including colorectal cancer (NCT01622543); prostate cancer (NCT01619813); head and neck squamous cell carcinoma (NCT01166542); pancreatic adenocarcinoma (NCT00998322); and non-small cell lung cancer (NSCLC) (NCT-00861627). enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), a variant of respiratory enteric orphan virus that does not replicate in non-human ovarian cells (NCT02028117); an adenovirus engineered to express full-length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein in metastatic or advanced epithelial tumors, such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); melanoma (NCT03003676); and ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF in peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831). Vaccinia viruses engineered to express beta-galactosidase (beta-gal) / beta-glucuronidase or beta-gal / human sodium iodide symporter (hNIS) have been studied in fallopian tube and ovarian cancers (NCT02759588), respectively; or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, in bladder cancer (NCT02365818).

[0319] In some embodiments, the immuno-oncology agents include JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which can convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapeutics targeting intractable RAS mutations; and TILT-123 (TILT Biotherapeutics, i.e., an engineered adenovirus designated Ad5 / 3-E2F-delta24-hTNFα-IRES-hIL20; and VSV-GP (ViraTherapeutics, i.e., a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express an antigen designed to enhance antigen-specific CD8+ T cell responses).

[0320] In some embodiments, the immuno-oncology agent is a T cell engineered to express a chimeric antigen receptor, or CAR. Such T cells engineered to express a chimeric antigen receptor are called CAR-T cells.

[0321] CARs are constructed that consist of a binding domain, which can be derived from a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for a cell surface antigen, which is a natural ligand, and fused to an endodomain, which is the functional end of a T cell receptor (TCR), such as the CD3-zeta signaling domain derived from the TCR, that can generate an activation signal in T lymphocytes. Upon binding to an antigen, such a CAR links to the endogenous signaling pathway of the effector cell and generates an activation signal similar to that initiated by the TCR complex.

[0322] For example, in some embodiments, the CAR-T cells are one of those described in U.S. Pat. No. 8,906,682 (une et al.; incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to include an extracellular domain having an antigen-binding domain (e.g., a domain that binds to CD19) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3-zeta). When expressed in T cells, CARs can redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Over 200 clinical trials are currently underway using CAR-T cells in a wide range of indications. [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1]

[0323] In some embodiments, the immunostimulatory agent is an activator of retinoic acid receptor-related orphan receptor gamma (RORγt). RORγt is a transcription factor that plays a key role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as in the differentiation of IL-17-expressing innate immune cell subsets, such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT02929862).

[0324] In some embodiments, the immunostimulatory agent is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLRs include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG currently being tested in follicular B-cell and other lymphomas (NCT02254772). Agonists or activators of TLR8 that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals), currently being tested in squamous cell carcinoma of the head and neck (NCT02124850) and ovarian cancer (NCT02431559).

[0325] Other immuno-oncology agents that can be used in the present invention include urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; valilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirilumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0326] In some embodiments, the immunostimulatory agent is selected from elotuzumab, mifamurtide, an agonist or activator of a toll-like receptor, or an activator of RORγt.

[0327] In some embodiments, the immune stimulatory therapeutic agent is recombinant human interleukin-15 (rhIL-15). rhIL-15 is being clinically tested as a treatment for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888), and leukemia (NCT02689453). In some embodiments, the immune stimulatory agent is recombinant human interleukin-12 (rhIL-12). In some embodiments, the IL-15-based immunotherapeutic agent is heterodimeric IL-15 (hetIL-15, Novartis / Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed to the soluble IL-15 binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), which is being tested in Phase 1 clinical trials for melanoma, renal cell carcinoma, non-small cell lung cancer, and head and neck squamous cell carcinoma (NCT02452268). In some embodiments, the recombinant human interleukin-12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724, or NCT02542124.

[0328] In some embodiments, the immuno-oncology agent is described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pages 603-622, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is selected from the examples listed in Table 1 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule that targets an immune-oncology target selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule drug selected from those listed in Table 2 of Jerry L. Adams et al.

[0329] In some embodiments, the immuno-oncology agent is selected from the small molecule immuno-oncology agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pages 319-329, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is an agent that targets a pathway as described in Peter L. Toogood.

[0330] In some embodiments, the immuno-oncology agent is selected from those described in Sandra L. Ross et al., "Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing," PLoS ONE 12(8): e0183390, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is a bispecific T cell engager (BiTE®) antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates T cells. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates the T cell, causing it to release cytokines that induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates the T cell, causing it to induce bystander cell lysis. In some embodiments, the bystander cells are within a solid tumor. In some embodiments, the bystander cells to be lysed are in proximity to the BiTE®-activated T cells. In some embodiments, the bystander cells comprise tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, the bystander cells comprise EGFR-negative cancer cells. In some embodiments, the immuno-oncology agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immuno-oncology agent is ex vivo expanded tumor-infiltrating T cells.In some embodiments, the immuno-oncology agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly connects T cells to tumor-associated surface antigens (TAA).

[0331] Exemplary Immune Checkpoint Inhibitors In some embodiments, the immuno-oncology agent is an immune checkpoint inhibitor described herein.

[0332] The term "checkpoint inhibitor" used herein refers to a drug that is useful for preventing cancer cells from evading the patient's immune system.One of the main mechanisms of anti-tumor immune destruction is known as "T cell exhaustion", which is caused by long-term exposure to antigen, which leads to the upregulation of inhibitory receptors.These inhibitory receptors function as immune checkpoints to prevent indiscriminate immune responses.

[0333] PD-1 and its co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin and mucin domain-3 (Tim-3), and lymphocyte-activation gene-3 (Lag-3; CD223), are often referred to as checkpoint regulators. They function as molecular "gatekeepers" that allow extracellular signals to dictate whether cell cycle progression and other intracellular signaling processes should proceed.

[0334] In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1, which binds to the programmed cell death 1 receptor (PD-1) and prevents the receptor from binding to the inhibitory ligand PDL-1, thus abolishing the tumor's ability to suppress the host's anti-tumor immune response.

[0335] In some embodiments, the checkpoint inhibitor is a biotherapeutic or small molecule. In some embodiments, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. The checkpoint inhibitor inhibits a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or a combination thereof. In some embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, a ligand of the B-7 family, or a combination thereof. In some embodiments, the checkpoint inhibitor is an immunostimulant, a T cell growth factor, an interleukin, an antibody, a vaccine, or a combination thereof. In some embodiments, the interleukin is IL-7 or IL-15. In some embodiments, the interleukin is glycosylated IL-7. In a further aspect, the vaccine is a dendritic cell (DC) vaccine.

[0336] Checkpoint inhibitors include any agent that blocks or inhibits an inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors can include small molecule inhibitors, or can include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (a member of the CD2 family of molecules expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (a PD-1 blocker), nivolumab (an anti-PD1 antibody), CT-011 (an anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A (an anti-PDL1 antibody), MSB0010718C (an anti-PDL1 antibody), and ipilimumab (an anti-CTLA-4 checkpoint inhibitor).Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0337] In certain embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (OPDIVO®), ipilimumab (YERVOY®), and pembrolizumab (KEYTRUDA®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, OPDIVO®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, KEYTRUDA®, Merck); ipilimumab (anti-CTLA-4 antibody, YERVOY®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, IMFINZI®, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, TECENTRIQ®, Genentech).

[0338] In some embodiments, the checkpoint inhibitor is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, rilumab, IPH2101, pembrolizumab (KEYTRUDA®), and tremelimumab.

[0339] In some embodiments, the immune checkpoint inhibitor is REGN2810 (Regeneron), an anti-PD-1 antibody that has been studied in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), an antibody that binds to PD-1, also known as CT-011, in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab (BAVENCIO®, Pfizer / Merck), in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer. KGaA), also known as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody; and PDR001 (Novartis), an inhibitory antibody that binds to PD-1, which is in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 that has been studied in clinical trials for multiple indications, including mesothelioma, colorectal cancer, renal cancer, breast cancer, lung and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic cancer in the liver, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody being studied in a Phase 1 clinical trial for advanced solid tumors (NCT02694822).

[0340] In some embodiments, the checkpoint inhibitor is an inhibitor of T-cell immunoglobulin mucin-containing protein-3 (TIM-3). TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody currently being tested in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody currently being tested in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody currently being tested in advanced malignancies (NCT02608268).

[0341] In some embodiments, the checkpoint inhibitor is an inhibitor of T cell immunoreceptor with Ig and ITIM domains, i.e., TIGIT, an immunoreceptor on certain T cells and NK cells. TIGIT inhibitors that can be used in the present invention include BMS-986207 (Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428).

[0342] In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene 3 (LAG-3). LAG-3 inhibitors that can be used in the present invention include BMS-986016, REGN3767, and IMP321. BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is currently being tested in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody and is currently being tested in malignant tumors (NCT03005782). IMP321 (Immutep SA), a LAG-3-Ig fusion protein, is currently being tested in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[0343] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists currently being tested in clinical trials include PF-04518600 / PF-8600 (Pfizer), an agonistic anti-OX40 antibody, in metastatic kidney cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonistic anti-OX40 antibody, in a Phase 1 cancer clinical trial (NCT02528357); and in advanced solid tumors (NCT02318394 and NCT02705482). These include MEDI0562 (Medimmune / AstraZeneca), an agonistic anti-OX40 antibody; MEDI6469, an agonistic anti-OX40 antibody (Medimmune / AstraZeneca), in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb), an agonistic anti-OX40 antibody, in advanced cancers (NCT02737475).

[0344] Checkpoint inhibitors that can be used in the present invention include CD137 (also known as 4-1BB) agonists. CD137 agonists currently being tested in clinical trials include utomilumab (PF-05082566, Pfizer), an agonistic anti-CD137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and neoplasms (NCT02554812 and NCT05082566); urelumab (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD137 antibody, in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981); and CTX-471 (Compass Therapeutics), an agonistic anti-CD137 antibody, in metastatic or locally advanced malignancies (NCT03881488).

[0345] Checkpoint inhibitors that can be used in the present invention include CD27 agonists.CD27 agonists currently being tested in clinical trials include varlilumab (CDX-1127, Celldex Therapeutics), an agonistic anti-CD27 antibody, in squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0346] Checkpoint inhibitors that can be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists currently being tested in clinical trials include TRX518 (Leap) in malignant melanoma and other malignant solid tumors (NCT01239134 and NCT02628574). GWN323 (Novartis), an agonistic anti-GITR antibody, in solid tumors and lymphomas (NCT02740270); INCAGN01876 (Incyte / Agenus), an agonistic anti-GITR antibody, in advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonistic anti-GITR antibody, in solid tumors (NCT02132754), and MEDI1873 (Medimmune / AstraZeneca), an agonistic hexameric GITR ligand molecule with a human IgG1 Fc domain, in advanced solid tumors (NCT02583165).

[0347] The checkpoint inhibitor that can be used in the present invention includes inducible T cell costimulator (ICOS, also known as CD278) agonist.The ICOS agonist currently being tested in clinical trials includes MEDI-570 (Medimmune), an agonistic anti-ICOS antibody, in lymphoma (NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody, in phase 1 trial (NCT02723955); JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody, in phase 1 trial (NCT02904226).

[0348] Checkpoint inhibitors that can be used in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors currently being tested in clinical trials include lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody, in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma), in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to the long cytoplasmic tail three domain (KIR3DL2), in lymphoma (NCT02593045).

[0349] Checkpoint inhibitors that can be used in the present invention include CD47 inhibitors of the interaction between CD47 and signal-regulatory protein alpha (SIRPa). CD47 / SIRPa inhibitors currently being tested in clinical trials include ALX-148 (Alexo Therapeutics), an antagonistic variant of (SIRPa) that binds to CD47 and prevents CD47 / SIRPa-mediated signaling, in a Phase 1 trial (NCT03013218); and TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein generated by linking the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1, which binds to human CD47 and prevents human CD47 from binding to its "do not eat" domain, in Phase 1 clinical trials (NCT02890368 and NCT02663518). These include CC-90002 (Celgene), an anti-CD47 antibody, in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.), in colorectal neoplasia and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0350] Checkpoint inhibitors that can be used in the present invention include CD73 inhibitors. CD73 inhibitors currently being tested in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody, in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody, in solid tumors (NCT02754141).

[0351] Checkpoint inhibitors that can be used in the present invention include agonists of the stimulator of interferon genes protein (STING, also known as transmembrane protein 173, or TMEM173). STING agonists currently being tested in clinical trials include MK-1454 (Merck), an agonistic synthetic cyclic dinucleotide, in lymphoma (NCT03010176); and ADU-S100 (MIW815, Aduro Biotech / Novartis), an agonistic synthetic cyclic dinucleotide, in Phase 1 trials (NCT02675439 and NCT03172936).

[0352] Checkpoint inhibitors that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors currently being tested in clinical trials include pexidartinib (PLX3397, Plexxikon), a CSF1R small molecule inhibitor, in colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710), as well as melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST), and ovarian cancer (NCT02452424); and pexidartinib (PLX3397, Plexxikon), a CSF1R small molecule inhibitor, in pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (SCD03101254). These include IMC-CS4 (LY3022855, Lilly), an anti-CSF-1R antibody, in advanced solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide, Novartis), an orally available inhibitor of CSF1R, in advanced solid tumors (NCT02829723).

[0353] Checkpoint inhibitors that can be used in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors currently being tested in clinical trials include monalizumab (IPH2201, Innate Pharma), an anti-NKG2A antibody, in head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0354] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab. [Example]

[0355] The following examples are intended to illustrate the invention and should not be construed as limiting thereof. Unless otherwise specified, all amino acids were used in the L-configuration. [Table 1]

[0356] Example 1. Multi-tumor survey of Nectin-4 expression to guide BT8009 indication selection 1.1 Nectin-4 IHC protocol Reagents / probes / antibodies [Table 2] [Table 3] procedure 1. Fix and embed tissue, cut sections and mount them on positively charged slides, and deparaffinize and rehydrate sections according to standard methods. 2. Load the sample into the Leica Bond III 3. Incubate with Bond Dewax solution for 2 minutes at room temperature. 4. Incubate with alcohol for 2 minutes at room temperature. 5. Rinse with Bond Wash Solution 6. Incubate with Bond Epitope Retrieval Solution 1 at 100°C for 20 minutes. 7. Rinse with Bond Wash Solution 8. Block with Bond Peroxide Block for 5 minutes at ambient temperature. 9. Rinse with Bond Wash Solution 10. Block with Dako Protein Block for 10 minutes at ambient temperature. 11. Clean with Bond Wash Solution 12. Incubate with anti-nectin-4 antibody (10 μg / ml) diluted in Dako Background Reducing Diluent for 30 minutes at ambient temperature. 13. Rinse with Bond Wash Solution 14. Incubate with Post Primary for 15 minutes at room temperature. 15. Rinse with Bond Wash Solution 16. Incubate with Bond Polymer for 15 minutes at room temperature. 17. Rinse with Bond Wash Solution 18. Incubate with Bond Mixed DAB Refine Reagent (Part 1) followed by Bond Mixed DAB Refine Reagent (Part 2) for 10 minutes at ambient temperature. 19. Rinse with DI water 20. Incubate with Bond hematoxylin for 5 minutes at ambient temperature. 21. Rinse with DI water 22. Rinse with Bond Wash Solution 23. Rinse with DI water 24. Remove, dehydrate in graded alcohols, and clear in xylene for 7 minutes at ambient temperature. 25. Coverslip

[0357] Nectin-4 IHC scoring Score the Nectin-4 staining results using the H-score method (defined as the sum of the product of the percent of cells times their staining intensity, on a scale of 0 to 3, where 0 is negative and 3 is strong staining). Separate H-scores for the cell membrane and cytoplasm can be generated to distinguish between the two compartments.

[0358] 1.2 Results A clinical-grade nectin-4 IHC assay was developed on the Leica platform using a rabbit monoclonal α-nectin-4 primary antibody (Abcam, Burlingame, CA) and the Bond Polymer Refine detection kit. Using the tumor microenvironment assessed in a subset of samples, TMAs from cancer types reported to have high nectin-4 expression, including esophageal, pancreatic, bladder, head and neck, gastric, non-small cell lung, breast, and ovarian cancers, were stained and nectin-4 levels were manually scored. Nectin-4 H scores (staining intensity on a scale of 0–3× percent of positive tumor cells) were generated independently by a pathologist for the tumor cell membrane and tumor cytoplasm. An H score of ≥100 for the tumor membrane or cytoplasm was considered positive. [Table 4]

[0359] In all indications tested, higher levels of Nectin-4 positivity were observed in the tumor cytoplasm compared with the tumor membrane. Breast and bladder cancers had the highest frequency of Nectin-4 positivity. Subtype analysis of breast cancer confirmed that Nectin-4 expression was higher in hormone receptor-negative tumors and human epidermal growth factor receptor 2-positive tumors.

[0360] Conclusions: The frequency of Nectin-4 expression measured by IHC across multiple tumor types can guide the clinical strategy of the BT8009 program.

[0361] Example 2. Molecular-based enrichment strategy for the nectin-4-targeting bicyclic toxin conjugate BT8009 Materials: Tumor cores were obtained from US Biomax (Rockville, MD) and used for TMA construction and subsequent IHC staining, as well as DNA extraction and subsequent whole-exome sequencing. The TMA (BR1301: https: / / www.biomax.us / tissue-arrays / Breast / BR1301) consisted of 120 TNBC cases and controls (TNBC controls: two each of ER+, PR+, and HER2+, and Nectin-4 protein expression controls: two spleen tissues and two skin tissues).

[0362] Nectin-4 protein expression: IHC was performed using the Nectin-4 IHC assay. Tumor membrane and cytoplasmic H scores were determined by a pathologist.

[0363] Whole-exome sequencing: Whole-exome sequencing was performed on approximately 110 TNBC samples. DNA and RNA were co-extracted from FFPE tissue using Qiagen's AllPrep DNA / RNA FFPE kit. DNA QC, whole-exome library construction (including library preparation and hybrid capture), and exome sequence generation were performed on the following samples: All samples that passed DNA QC and sequence metric cutoffs were advanced to DNA variant analysis.

[0364] DNA variant analysis: Somatic analysis of tumor only was performed against the normal panel using the GATK4 MuTect2 (SNV / Indels) and GATK4 CNV pipelines.

[0365] Whole-exome sequencing data preprocessing: A total of 100 copy number segmentation files were pre-annotated using Oncotator. The annotated copy number segmentation files identified the genomic location, copy number call (diploid, amplification, or deletion), average log2-transformed copy ratio, and genes located within regions of each contiguous chromosomal segment with consistent ploidy. These segment data were then expanded into gene-level data, in which genes were assigned the allele frequency of their parent chromosomal segment. Genes with inconsistent ploidy, as indicated by their presence in multiple chromosomal segments, were excluded from the analysis.

[0366] The goal of this experiment was to identify routinely measured molecular surrogates for tumor Nectin-4 protein expression (i.e., somatic mutations or gene amplifications) that could potentially increase patient screening frequency, yield, and likelihood of response to BT8009. Here, we show that Nectin-4 copy number is associated with Nectin-4 mRNA expression in nine TCGA cancer indications, including breast, bladder, and lung cancer. Furthermore, across >30 TCGA cancer indications, Nectin-4 copy number is highly correlated with SDHC copy number (these two genes are separated by approximately 225 kb on 1q23), which is included in multiple commercially available NGS panels. Collectively, these results suggest that the presence of SDHC amplification can be used as an enrichment tool to identify patients with Nectin-4-positive tumors. One hundred TNBC human tumor samples were tested to determine Nectin-4 and SDHC copy number, as well as Nectin-4 protein expression status by IHC.

[0367] Results: We observed that SDHC and Nectin-4 copy numbers were highly positively correlated (R2 = 0.93). Furthermore, all (N = 22) TNBC tumors with Nectin-4 copy numbers ≥ 3 were identified as positive for Nectin-4 protein expression and were above the H-score threshold required for enrollment in BT8009-100. In contrast, the subset of tumors diploid for Nectin-4 / SDHC (n = 30) contained both Nectin-4-positive and -negative tumors. This suggests that the presence of SDHC / Nectin-4 amplification can be used to increase the likelihood of identifying Nectin-4-expressing tumors.

[0368] Conclusion: Existing patient NGS data with SDHC amplification was shown to be useful for identifying patients with Nectin-4-high expressing tumors via IHC.

[0369] Example 3. MultiOmyx™ Hyperplex Immunofluorescence Assay Using MultiOmyx™ technology, we assessed the expression of a panel of 19 biomarkers, including EphA2, Nectin-4, CD137, CD19, CD3, CD4, CD8, FOXP3, CD69, CD45RO, CD56, CD68, CD11b, Granzyme B, PD-1, PD-L1, HLA-ABC, Ki67, and the tumor segmentation marker PanCK, in head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), breast cancer, and bladder cancer FFPE samples. Each FFPE slide was presented to a NeoGenomics pathologist for tissue annotation and selection. Tumor-associated areas selected by the pathologist were used for staining and analysis. Staining was performed using a single 4µM FFPE slide. Within each staining round, up to two markers were recognized using a combination of two cyanine dye-labeled (Cy3, Cy5) antibodies. The staining signal was then imaged, followed by deactivation of the new dye, allowing for repeated rounds of staining. A proprietary deep learning-based workflow was applied to identify individual cells, perform cell classification for all individual markers, and identify tissues and tumor regions for analysis. Individual cell and region classification results were combined to generate co-expression summaries and calculate spatial distribution statistics for phenotypes of interest.

[0370] The MultiOmyx™ Hyperplex immunofluorescence assay was developed to simultaneously quantify the presence of Nectin-4 and CD137-positive cells and the topography of these cell types in 15 primary human tumor samples in situ (shown in Figure 10B). The frequency of Nectin-4 and CD137-positive cells detected in each tumor sample indicated the presence of significant CD137+ immune cell infiltration within Nectin-4-positive NSCLC, HNSCC, and bladder cancer samples. Spatial profiling analysis further revealed that CD137+ immune infiltrates were detected within the tumor stroma, with some of these penetrating deep into the tumor core and in closest proximity to Nectin-4-positive tumor cells (Figure 10C). More detailed profiling revealed that the CD137-expressing immune cells detected in all tumor types analyzed were primarily composed of CD4+ and CD8+ T cells, as well as CD68+ macrophages (Figure 10D). Although the effects of CD137 TCA may not be restricted to T cells, these data indicate that at least CD137-bearing T cells are more likely to contact Nectin-4-bearing tumor cells. Thus, these observations support the development of tumor-targeted CD137 agonists for the treatment of Nectin-4-positive human cancers.

[0371] While numerous embodiments of the present invention have been described, it will be apparent that the examples may be modified to provide other embodiments that utilize the compounds and methods of the present invention. It will therefore be understood that the scope of the present invention is to be defined by the application and claims, rather than by the specific embodiments that have been represented by way of example.

Claims

1. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, the method comprising measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting a patient having an elevated Nectin-4 DNA copy number in the tumor tissue.

2. 2. The method of claim 1, wherein the step of measuring the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) comprises using next-generation sequencing (NGS) technology or sequence-based sequence capture.

3. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, comprising measuring the Nectin-4 DNA copy number of the patient using next-generation sequencing (NGS) technology or sequence-based sequence capture, and selecting a patient having an elevated Nectin-4 DNA copy number.

4. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, the method comprising measuring the SDHC DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting a patient having an elevated SDHC DNA copy number in the tumor tissue.

5. 5. The method of claim 4, wherein the step of measuring the SDHC DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) comprises using next-generation sequencing (NGS) technology or sequence-based sequence capture.

6. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, comprising measuring the SDHC DNA copy number of the patient using next-generation sequencing (NGS) technology or sequence-based sequence capture, and selecting a patient having an elevated SDHC DNA copy number.

7. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, the method comprising measuring the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting a patient having an elevated DDR2 DNA copy number in the tumor tissue.

8. 8. The method of claim 7, wherein the step of measuring the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) comprises using next generation sequencing (NGS) technology or sequence-based sequence capture.

9. A method for identifying or selecting a patient having an elevated Nectin-4 protein level and / or RNA expression level in tumor tissue, the method comprising measuring the DDR2 DNA copy number of the patient using next-generation sequencing (NGS) technology or sequence-based sequence capture, and selecting a patient having an elevated DDR2 DNA copy number.

10. 10. The method of any one of claims 1 to 9, wherein the patient has pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or ovarian cancer.

11. The method of any one of claims 1 to 3, wherein the selected patient has a Nectin-4 Log2 (CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more.

12. 7. The method of any one of claims 4 to 6, wherein the selected patient has an SDHC Log2 (CN ratio) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, or about 1.0 or greater.

13. 10. The method of any one of claims 7 to 9, wherein the selected patient has a DDR2 Log2 (Centricity Ratio) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, or about 1.0 or greater.

14. A method for treating cancer in a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, the method comprising administering to the patient a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

15. The method of claim 14, wherein a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue refers to a patient having a Nectin-4 Log2 (CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more, measured in the patient's tumor tissue or circulating tumor DNA (ctDNA).

16. The method of claim 14, wherein a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue refers to a patient having an SDHC Log2 (CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more, measured in the patient's tumor tissue or circulating tumor DNA (ctDNA).

17. The method of claim 14, wherein a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue refers to a patient having a DDR2 Log2 (CN ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more, measured in the patient's tumor tissue or circulating tumor DNA (ctDNA).

18. A method of treating cancer in a patient, comprising selecting a patient having a Nectin-4 Log2 (C-N ratio) of about 0.1 or more, about 0.2 or more, about 0.3 or more, about 0.4 or more, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, about 0.9 or more, or about 1.0 or more in tumor tissue or circulating tumor DNA (ctDNA) as determined by next-generation sequencing (NGS) technology or sequence-based sequence capture, and administering to the patient a Nectin-4-specific bicyclic toxin conjugate or bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

19. A method of treating cancer in a patient, comprising selecting a patient having an SDHC Log2 (C-N ratio) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, or about 1.0 or greater in tumor tissue or circulating tumor DNA (ctDNA) as determined by next-generation sequencing (NGS) technology or sequence-based sequence capture, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4 or a bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

20. A method of treating cancer in a patient, comprising selecting a patient having a DDR2 Log2 (C-N ratio) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, or about 1.0 or greater in tumor tissue or circulating tumor DNA (ctDNA) as determined by next-generation sequencing (NGS) technology or sequence-based sequence capture, and administering to the patient a bicyclic toxin conjugate specific for Nectin-4 or a bicyclic TICA, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

21. 21. The method of any one of claims 14 to 20, wherein the cancer is pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or ovarian cancer.

22. The method of any one of claims 14 to 21, further comprising administering an immuno-oncology agent.

23. 23. The method of claim 22, wherein the immuno-oncology agent is a PD-1 antagonist.

24. 24. The method of claim 23, wherein the PD-1 antagonist is an antagonistic PD-1 antibody.

25. 25. The method of claim 24, wherein the antagonistic PD-1 antibody is selected from nivolumab, pembrolizumab, MEDI-0680, pidilizumab, AMP-224, atezolizumab, durvalumab, BMS-936559, avelumab, or cemiplimab.

26. The method of any one of claims 14 to 25, wherein the bicyclic toxin conjugate specific for Nectin-4 is BT8009 or the bicyclic TICA is BT7480.

27. A method for identifying or selecting a patient in whom Nectin-4 protein level and / or RNA expression level in tumor tissue is elevated, the method comprising measuring the Nectin-4 protein level in the patient's tumor tissue, and selecting a patient in whom the Nectin-4 protein level in the tumor tissue is elevated.

28. 28. The method of claim 27, wherein said step of measuring Nectin-4 protein levels in the patient's tumor tissue comprises using a Nectin-4 multiplexed immunofluorescence (mIF) assay.

29. A method for identifying or selecting a patient whose tumor tissue has elevated Nectin-4 protein levels and / or RNA expression levels, the method comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4 mIF assay, and selecting patients who stain positive in the Nectin-4 mIF assay.

30. 30. The method of any one of claims 27-29, wherein the patient has pancreatic cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or ovarian cancer.

31. The method of claim 28 or 29, wherein the Nectin-4 mIF assay uses a rabbit monoclonal α-Nectin-4 primary antibody that selectively binds to the extracellular domain (ECD) of Nectin-4.

32. The method of claim 31, wherein the rabbit monoclonal α-nectin-4 primary antibody that selectively binds to the extracellular domain (ECD) of nectin-4 is rabbit monoclonal α-nectin-4 primary antibody YMW-1-58.

33. The method of claim 29, wherein positive staining in the Nectin-4 mIF assay refers to an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in the tumor tissue section in the Nectin-4 mIF assay.

34. The method of claim 29, wherein positive staining in the Nectin-4 mIF assay refers to an H-score of tumor cell membranes in tumor tissue sections in the Nectin-4 mIF assay of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more.

35. The method of claim 29, wherein positive staining in the Nectin-4 mIF assay refers to an H-score of tumor cell cytoplasm of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in the tumor tissue section in the Nectin-4 mIF assay.

36. A method for treating cancer in a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, the method comprising administering to the patient a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

37. The method of claim 36, wherein a patient having elevated Nectin-4 protein and / or RNA expression levels in tumor tissue refers to a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay.

38. A method for treating cancer in a patient, comprising selecting a patient having an H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, and administering to the patient a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

39. 39. The method of any one of claims 36 to 38, wherein the cancer is pancreatic cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or ovarian cancer.

40. The method of claim 37 or 38, wherein the Nectin-4 mIF assay uses a rabbit monoclonal α-Nectin-4 primary antibody that selectively binds to the extracellular domain (ECD) of Nectin-4.

41. The method of claim 40, wherein the rabbit monoclonal α-nectin-4 primary antibody that selectively binds to the extracellular domain (ECD) of nectin-4 is rabbit monoclonal α-nectin-4 primary antibody YMW-1-58.

42. 39. The method of claim 38, wherein the H-score refers to a tumor cell membrane H-score or a tumor cell cytoplasm H-score.

43. A method for treating cancer in a patient, comprising selecting a patient having a tumor cell membrane H-score of about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, or about 150 or more in a tumor tissue section in a Nectin-4 mIF assay, and administering BT8009 or BT7480, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to the patient.

44. 44. The method of claim 43, wherein the cancer is pancreatic cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or ovarian cancer.

45. 44. The method of claim 43, wherein the Nectin-4 mIF assay uses a rabbit monoclonal α-Nectin-4 primary antibody that selectively binds to the extracellular domain (ECD).

46. The method of claim 45, wherein the rabbit monoclonal α-nectin-4 primary antibody that selectively binds to the extracellular domain (ECD) of nectin-4 is rabbit monoclonal α-nectin-4 primary antibody YMW-1-58.

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