Bicyclic conjugates specific for nectin-4 and uses thereof

Abstract

This invention relates to bicyclic toxin conjugates or bicyclic tumor-targeting immune agonists that are specific to Nectin-4, or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof, and their uses.

Description

Technical Field

[0001] This invention relates to bicyclic toxin conjugates or bicyclic tumor-targeting immune agonists (TICAs) that are specific to Nectin-4, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, and their use for the prevention or treatment of diseases, conditions, or disorders characterized by overexpression of Nectin-4 in diseased tissues such as tumor tissues. Background Technology

[0002] The presence of tumor gene amplification is associated with the upregulation of related encoded proteins, and the use of tests to determine the presence or absence of gene amplification as a predictor of therapeutic response has been well-established in the treatment of solid tumors. Amplification of ERBB2 (HER2), MET, and FGFR has all been shown to predict responses to therapeutic agents 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 various tumor types {Challita-Eid et al., Cancer Research, 2016}, and it has been associated with potential Nectin-4 gene amplification in breast cancer {N Pavlova et al., Elife, 2013}. Analysis of TCGA data suggests that Nectin-4 amplification occurs in other indications and therefore may represent a way to identify tumors with high Nectin-4 expression in indications that can respond to Nectin-4 targeted therapies such as BT8009.

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

[0004] Due to their cyclic conformation, macrocyclic peptides are less flexible than linear peptides, resulting in less entropy loss and higher binding affinity when binding to a target. This reduced flexibility also leads to locking in a target-specific conformation, increasing binding specificity compared to linear peptides. This effect has been demonstrated by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity relative to other MMPs when its ring is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties achieved through macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0005] Different research teams have previously linked peptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings onto synthetic scaffolds for structural simulation of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for generating candidate drug compounds, wherein the compounds are generated by linking cysteine-containing peptides to molecular scaffolds such as TATA(1,1′,1″-(1,3,5-triazine-1,3,5-triyl)tripropyl-2-en-1-one, Heinis et al., Angew Chem, Int Ed. 2014; 53: 1602-1606).

[0006] Combinatorial methods based on phage display have been developed to generate large bicyclic peptide libraries and screen them for target molecules (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7, and WO 2009 / 098450). In short, a combinatorial library of linear peptides containing two regions (Cys-(Xaa)6-Cys-(Xaa)6-Cys) with three cysteine ​​residues and six random amino acids is displayed on a phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. Attached Figure Description

[0007] Figure 1 The results showed that Nectin-4 gain and amplification were statistically significantly associated with increased gene expression in various cancers, such as sarcoma, endometrial cancer, pancreatic cancer, lung adenocarcinoma, breast cancer, squamous cell carcinoma of the lung, head and neck cancer, cervical cancer, and bladder cancer.

[0008] Figure 2A The genes flanking Nectin-4 in the MSK-affected combination panel and the base drug combination were shown. Figure 2B The gene names, descriptions, genomic locations, and distances to Nectin-4 for SDHC and DDR2 are displayed.

[0009] Figure 3A This demonstrates a 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 separately for individual tumors by tumor group.

[0010] Figure 3B This demonstrates a 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 separately for individual tumors by tumor group.

[0011] Figure 4 Candidate alternative markers for identifying Nectin-4 protein expression = SDHC (or DDR2) amplification are shown. SDHC amplification can be used as an alternative marker to identify tumors with high Nectin-4 expression, which can be used to predict responses to Nectin-4 bicyclic toxin conjugates (BTC).

[0012] Figure 5A The analysis shows the Nectin-4 DNA copy number and protein expression in the membrane in TNBC samples. Figure 5B The analysis shows the Nectin-4 DNA copy number and protein expression in the cytoplasm in TNBC samples. Figure 5C The analysis shows the Nectin-4 DNA copy number and protein expression in the membrane and cytoplasm in TNBC samples. Note that a combined H score of ≥100 for all tumor nuclei is required for a Log2(CN ratio) ≥ approximately 0.6. Figure 5D The results show the 100% positive predictive value when CN≥3 is used to determine a tumor membrane + cytoplasmic H score ≥100. Figure 5A , Figure 5B and Figure 5CThe X-axis of each graph is Nectin-4Log2 (CN ratio), and the dashed lines in both graphs show the H score and the quartiles of the Nectin-4 DNA copy number.

[0013] Figure 6A The Nectin-4 DNA copy number gain was shown to be correlated with a rightward shift in the combined membrane and cytoplasmic H scores. Figure 6B The relationship between the H-score cutoff and copy number calls is shown, as determined by the GATK pipeline (https: / / gatk.broadinstitute.org / hc / en-us). (H-score boundaries are included).

[0014] Figure 7A (Membrane + Cytoplasmic H Score) Figure 7B (Membrane H score) and Figure 7C (Cytoplasmic H score) shows the number of samples that meet the H score cutoff value as the Nectin-4 log2 copy number cutoff value increases. Both the H score and the log2 CN cutoff value are included. Figure 7D The results show TNBC samples at different membrane + cytoplasmic H score cutoffs (x-axis) under different Nectin-4log2 CN ratio thresholds.

[0015] Figure 8A This demonstrates that SDHC can be used as an alternative for Nectin-4 DNA copy number (and potential Nectin-4 protein expression).

[0016] Figure 8B This demonstrates that DDR2 can be used as an alternative for Nectin-4 DNA copy number (and potential Nectin-4 protein expression).

[0017] Figure 9 A model was constructed to depict the effect of enriching a patient population with higher membrane + cytoplasmic H scores using a Nectin-4 CN cutoff of log2 CN ratio ≥ 0.5. Methods: TNBC and TMA membrane + cytoplasmic H scores were resampled without replacement, using expected group sizes of 16 and 10,000 permutations, without using a log2 CN ratio cutoff of ≥ 0 or ≥ 0.5. H score intervals were inclusive on the left and exclusive on the right, except that the highest interval (bin) was inclusive on both sides.

[0018] Figure 10A The co-localization of Nectin-4-expressing tumor cells and CD137-expressing immune cells in human cancers was depicted. Transcripts co-expressed across tumor types were observed in TCGA.

[0019] Figure 10B MultiOmyx describes a technology that allows simultaneous probing of immune infiltration and spatial proteome distribution within human tumors. TM Imaging. A single ROI is shown from a representative HNSCC sample. T cells (CD3+, red), macrophages (CD68+, blue), NK cells (CD56+, green), and tumor cells (PanCK+, cyan) were detected throughout the tumor (top left). Instances of CD137+CD4+ and CD137+CD8+ T cells are shown and indicated by white and gray arrows, respectively (top right). Nectin-4 (red) and PanCK (blue) are co-expressed on tumor cells (bottom left). Tumor and stromal regions were identified using PanCK and DAPI masks, respectively (bottom right, indicated by red and blue, respectively).

[0020] Figure 10C The images depict tumor Nectin-4 expression (left) with total Nectin-4+ PanCK+ cells normalized to total cells and CD137+ immune infiltration (right) with total CD137+ cells normalized to total cells. Within each box, the horizontal line represents the mean of the five samples shown.

[0021] Figure 10D This study depicts a subgroup analysis of CD137+ immune infiltration across samples and within the stroma (left) and tumor (right) regions, including T cells (CD3+CD4+ and CD3+CD8+), macrophages (CD68+), NK cells (CD56+), and B cells (CD19+). Data are total cells for each phenotype, normalized to the total CD137+ cells detected in samples within each indication. Within each box, the horizontal line represents the mean of the five samples shown. Summary of the Invention

[0022] As described herein, the inventors have discovered a correlation between Nectin-4 protein expression levels and Nectin-4 DNA copy number in diseased tissues. Nectin-4 is overexpressed in many difficult-to-treat tumors such as NSCLC, TNBC, pancreatic cancer, ovarian cancer, gastric / upper gastrointestinal 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 bicyclic toxin conjugate specific to Nectin-4. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4 is BT8009.

[0023] Furthermore, to avoid being bound by any particular theory or mechanism, tumors with elevated Nectin-4 RNA expression and / or elevated Nectin-4 DNA copy numbers in diseased tissue may be more likely to benefit from treatment with a bicyclic toxin conjugate specific to Nectin-4. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4 is BT8009.

[0024] Furthermore, to avoid being 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 co-localized with CD137-expressing immune cells may be more likely to benefit from treatment with a bicyclic tumor-targeting immune agonist specific to Nectin-4. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4 is BT7480.

[0025] 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-associated protein 4, poliovirus receptor-associated 4, Ig superfamily receptor LNIR, PVRL4, Nectin 4, NECTIN4, and EDSS 1.

[0026] In one aspect, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring Nectin-4 protein levels in the patient's diseased tissue and selecting patients with elevated Nectin-4 protein levels in the diseased tissue.

[0027] In another aspect, this article provides a method for treating a patient with elevated levels of Nectin-4 protein in diseased tissue (e.g., as determined using the methods described herein), the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0028] In another aspect, the present invention provides a method for treating a patient’s disease, the method comprising selecting a patient with elevated levels of Nectin-4 protein in diseased tissue, for example, using the method described herein, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0029] In some embodiments, the disease is cancer, such as the cancer described herein. In some embodiments, the diseased tissue is tumor tissue. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4 is selected from those described herein, such as BT8009 or a pharmaceutically acceptable salt thereof.

[0030] In another aspect, this article provides a method for treating a patient with elevated levels of Nectin-4 protein in diseased tissue (e.g., as determined using the methods described herein), the method comprising administering to the patient a bicyclic tumor-targeting immune agonist specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0031] In another aspect, the present invention provides a method for treating a patient’s disease, the method comprising selecting a patient with elevated levels of Nectin-4 protein in diseased tissue, for example, using the method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0032] In some embodiments, the disease is cancer, such as the cancer described herein. In some embodiments, the diseased tissue is tumor tissue. In some embodiments, the bicyclic tumor-targeting immune agonist specific to Nectin-4 is selected from those described herein, such as BT7480 or a pharmaceutically acceptable salt thereof. Detailed Implementation

[0033] 1. General description of certain embodiments of the present invention:

[0034] Recently, it has become common practice to submit tumor tissues at the initial diagnosis or first recurrence of solid tumors for genetic and genomic characterization using gene combinations developed locally at academic research centers {Cheng et al., J Molecular Diagnostics, 2015; Rothwell et al., Nature Medicine 2019} or commercially available {Miler et al., JCO, 2013; Lanman et al., PLoS One 2015}. These combinations are typically configured to identify specific somatic mutations, gene fusions, and gene amplification events. For combinations containing Nectin-4, patients can be identified by amplification of Nectin-4, which may indicate that their tumors predispose to high levels of Nectin-4 and therefore may respond to BT8009. Furthermore, some combinations include genes on chromosome 1 that are sufficiently close to Nectin-4, which are often amplified together, and therefore the detection of SDHC (or DDR2) amplification can serve as a substitute for Nectin-4 amplification, regardless of whether Nectin-4 is included in the combination. Therefore, the object of the present invention is to enrich patients with high Nectin-4 expression as determined by IHC that is responsive to BT8009 using patient tumor molecular genetic data regarding Nectin-4, SDHC, or DDR2 status.

[0035] SDHC is also known by the following aliases, each of which is equivalent to SDHC: succinate dehydrogenase complex subunit C; CYB560; succinate dehydrogenase complex subunit C membrane intrinsic protein, 15kD; mitochondrial succinate dehydrogenase cytochrome B560 subunit; succinate-ubiquinone oxidoreductase cytochrome B large subunit; succinate dehydrogenase cytochrome B; cytochrome B large subunit; CYBL; SDH3; succinate dehydrogenase complex subunit C membrane intrinsic protein, 15kDa; succinate dehydrogenase complex subunit C membrane intrinsic protein 15kDa; succinate dehydrogenase membrane intrinsic subunit; succinate dehydrogenase cytochrome B560 subunit; cytochrome B large subunit of complex II; membrane intrinsic protein CII-3b; membrane intrinsic protein CII-3; QPs-1; PGL3; QPS1; SDHC; CybL; and QPs1.

[0036] DDR2 is also known by the following aliases, each of which is equivalent to DDR2: discoid domain receptor tyrosine kinase 2; TKT; discoid domain-containing receptor tyrosine kinase 2; discoid domain receptor family member 2; discoid domain-containing receptor 2; receptor protein-tyrosine kinase TKT; CD167 antigen-like family member B; tyrosine protein kinase TYRO10; discoid domain receptor 2; EC 2.7.10.1; NTRKR3; TYRO10; neurotrophic tyrosine kinase receptor-associated protein 3; neurotrophic tyrosine kinase receptor-associated protein 3; cell migration-inducing protein 20; migration-inducing gene 16 protein; hydroxyaryl protein kinase; CD167b antigen; EC 2.7.10; MIG20a; WRCN; and DDR2.

[0037] Next-generation sequencing (NGS) technology, also known as high-throughput sequencing, allows for sequencing of DNA and RNA much faster and cheaper than the previously used Sanger sequencing. In some implementations, NGS technology is Illumina (Solexa) sequencing, which simultaneously identifies DNA bases because each base emits a unique fluorescent signal and is added to the nucleic acid strand. In some implementations, NGS technology is Roche 454 sequencing, which is based on pyrosequencing, a technique that uses fluorescence to detect pyrosequencing release after nucleotides have been incorporated into the new DNA strand by polymerase. In some implementations, NGS technology is ion-flux:proton / PGM sequencing, which measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase.

[0038] Nectin-4, SDHC, and DDR2 DNA copy numbers can be measured using NGS techniques as described herein. In some embodiments, Nectin-4, SDHC, and / or DDR2 DNA copy numbers are measured by sequencing the entire genome. In some embodiments, Nectin-4, SDHC, and / or DDR2 DNA copy numbers are measured by sequencing the entire exome.

[0039] Array-based sequence capture can also be used to sequence large target DNA regions in a less strenuous and time-consuming manner. In some embodiments, array-based methods are used to measure the copy number of Nectin-4, SDHC, and / or DDR2 DNA. In some embodiments, array-based methods are sequence-based. In some embodiments, array-based methods are non-sequence-based.

[0040] Circulating tumor DNA (ctDNA) exists in the bloodstream and refers to DNA originating from cancer cells and tumors. Most DNA resides inside the cell nucleus. As a tumor grows, cells die and are replaced by new cells. Dead cells are broken down, and their contents, including DNA, are released into the bloodstream. ctDNA consists of small pieces of DNA, typically containing fewer than 200 building blocks (nucleotides). Detection of ctDNA can be used to detect and diagnose tumors; guide tumor-specific treatments; monitor treatment progress; and monitor asymptomatic periods (cancer remission).

[0041] Nectin-4 DNA amplification in tumor tissues has been measured by whole-exome sequencing. It has been found that Nectin-4 DNA amplification is correlated with higher levels of Nectin-4 protein expression and can accordingly indicate tumors with elevated Nectin-4 expression. Without being bound by any particular theory or mechanism, the 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 toxin conjugates specific to Nectin-4.

[0042] Furthermore, not wishing to be bound by any particular theory or mechanism, the inventors have determined that tumors with elevated Nectin-4 DNA copy numbers in diseased tissues may be more likely to benefit from treatment with a bicyclic tumor-targeting immune agonist specific to Nectin-4.

[0043] SDHC or DDR2 DNA amplification in tumor tissues has been measured by whole-exome sequencing. SDHC or DDR2 DNA amplification has been found to act as a substitute for Nectin-4 expression and, correspondingly, to indicate tumors with elevated Nectin-4 expression. Without being 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 bicyclic toxin conjugate specific to Nectin-4. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4 is BT8009.

[0044] Furthermore, to avoid being bound by any particular theory or mechanism, tumors with elevated SDHC or DDR2 DNA copy numbers in diseased tissue may be more likely to benefit from treatment with a bicyclic tumor-targeting immune agonist specific to Nectin-4. In some implementations, the bicyclic tumor-targeting immune agonist specific to Nectin-4 is BT7480.

[0045] Therefore, in one aspect, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring Nectin-4 protein levels in the diseased tissue of the patient and selecting patients with elevated Nectin-4 protein levels in the diseased tissue.

[0046] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring Nectin-4 mRNA levels in the patient's diseased tissue and selecting patients with elevated Nectin-4 mRNA levels in the diseased tissue.

[0047] For each tissue microarray (TMA) nucleus analyzed, the membrane H-score (maximum 300) was added to the cytoplasmic H-score (maximum 300), resulting in a maximum combined total H-score of 600. This method differs from the standard method for calculating the H-score of the entire sample (TMA nucleus), which includes both membrane and cytoplasmic staining, resulting in a maximum H-score of 300. When calculating the H-score by positive % x intensity, the standard method assumes that the cytoplasm has a larger surface area, thus giving it a greater weight than the membrane. Not bound by any particular theory, one benefit of adding membrane and cytoplasmic H-scores is that this gives equal weight to membrane and cytoplasmic staining (even if the membrane surface area is smaller than the cytoplasmic surface area), providing a more appropriate score for Nectin-4BTC such as BT8009, which is expected to bind membrane Nectin-4. Figure 5A The analysis showed a comparison of membrane H scores and Nectin-4 DNA copy numbers, and... Figure 5B Analysis of isolated cytoplasmic H score and Nectin-4 DNA copy number is shown. Figure 5C The analysis shows the Nectin-4 DNA copy number and protein expression in the membrane and cytoplasm in TNBC samples. Figure 5D This shows the 100% positive predictive value when using CN≥3 to determine a tumor membrane + cytoplasmic H score ≥100. Figure 5D The following values ​​are given for sensitivity, specificity, positive predictive value, and negative predictive value, using a cutoff of CN ≥ 3:

[0048] Sensitivity = 28.21% (22 / 78)

[0049] • Specificity = 100% (22 / 22)

[0050] • Positive predictive value = 100% (22 / 22)

[0051] Negative predictive value = 28.21% (22 / 78)

[0052] in addition, Figure 7B (Separated membrane H score) and Figure 7C (H score of isolated cytoplasm) shows the number of samples that meet the H score cutoff value as the Nectin-4 log2 copy number cutoff value increases.

[0053] In some embodiments, the present invention provides a method for identifying or selecting patients with 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 patients with elevated Nectin-4 DNA copy numbers in their diseased tissue.

[0054] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring the copy number of SDHC and / or DDR2 DNA in the patient's diseased tissue, and selecting patients with elevated copy numbers of SDHC and / or DDR2 DNA in the diseased tissue.

[0055] In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 2 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 3 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 4 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 5 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 6 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 7 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 8 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 9 or greater. In some embodiments, an increased Nectin-4 DNA copy number means a copy number of 10 or greater.

[0056] In some embodiments, a Nectin-4 DNA copy number of 2 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 3 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 4 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 5 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 6 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 7 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 8 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 9 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay. In some embodiments, a Nectin-4 DNA copy number of 10 or greater is associated with an H score of 100 or higher in the Nectin-4 IHC staining assay.

[0057] In some embodiments, an increased SDHC DNA copy number means a copy number of 2 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 3 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 4 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 5 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 6 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 7 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 8 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 9 or greater. In some embodiments, an increased SDHC DNA copy number means a copy number of 10 or greater.

[0058] In some embodiments, an SDHC DNA copy number of 2 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 3 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 4 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 5 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 6 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 7 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 8 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 9 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay. In some embodiments, an SDHC DNA copy number of 10 or greater is associated with an H score of 100 or higher in the SDHC IHC staining assay.

[0059] In some embodiments, an increased DDR2 DNA copy number means a copy number of 2 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 3 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 4 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 5 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 6 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 7 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 8 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 9 or greater. In some embodiments, an increased DDR2 DNA copy number means a copy number of 10 or greater.

[0060] In some embodiments, a DDR2 DNA copy number of 2 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 3 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 4 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 5 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 6 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 7 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 8 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 9 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay. In some embodiments, a DDR2 DNA copy number of 10 or greater is associated with an H score of 100 or higher in the DDR2 IHC staining assay.

[0061] In another aspect, this article provides a method for treating a patient with elevated levels of Nectin-4 protein in diseased tissue (e.g., identified as elevated levels of Nectin-4 protein in diseased tissue using the methods described herein), the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0062] In another aspect, the present invention provides a method for treating a patient’s disease, the method 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 the methods described herein), and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0063] In some embodiments, the present invention provides a method for treating a patient’s disease, the method comprising, for example, selecting a patient with elevated Nectin-4 mRNA levels, Nectin-4 DNA copy numbers, SDHC DNA copy numbers, and / or DDR2 DNA copy numbers in diseased tissue using the methods described herein, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0064] In another aspect, this article provides a method for treating a patient with elevated levels of Nectin-4 protein in diseased tissue (e.g., identified as elevated levels of Nectin-4 protein in diseased tissue using the methods described herein), the method comprising administering to the patient a bicyclic tumor-targeting immune agonist specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0065] In another aspect, the present invention provides a method for treating a patient’s disease, the method comprising selecting 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), and administering to the patient a bicyclic tumor-targeting immune agonist specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0066] In another aspect, the present invention provides a method for treating a patient’s disease, the method comprising, for example, selecting a patient with elevated Nectin-4 mRNA levels, Nectin-4 DNA copy number, SDHC DNA copy number and / or DDR2 DNA copy number 1 in diseased tissue using the methods described herein, and administering to the patient a bicyclic tumor-targeting immune agonist specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0067] In some implementations, elevated Nectin-4, SDHC, or DDR2 DNA copy numbers are used to identify patients who may have tumors with high Nectin-4 protein expression (as measured by IHC). Figure 7DThe red curve (log2 CN ratio ≥ 0.6) is shown to shift to the right relative to the blue curve (no copy number cutoff). This indicates that Nectin-4 / SDHC / DDR2 amplification can be used to identify and enrich patients with higher Nectin-4 H scores. Without being bound by any particular theory, this identification will enrich patients with a higher percentage of Nectin-4 protein expression (as determined by IHC). This will potentially reduce the screening failure frequency, as defined by (number of patients not meeting the desired H score cutoff) / (total number of patients screened).

[0068] This concept is also shown in Figure 9 In the modeling described, Figure 9 Enrichment of tumors with higher Nectin-4 protein expression was depicted by using a log2 CN ratio ≥ 0.5 compared to not using the log2 CN ratio cutoff (shown as an Allcomers plot). This shift can be seen when comparing the leftmost 0-100 H score range between the Allcomers plot (left) and the log2 CN ratio ≥ 0.5 plot (right).

[0069] CD137 (4-1BB) is an immune co-stimulatory receptor with high therapeutic potential in cancer. Following disappointing initial clinical results from agonistic anti-CD137 antibodies, a new generation of systemic and targeted CD137 agonists is entering clinical development. Tumor target-dependent CD137 agonists have been developed using novel chemical approaches based on fully synthetic restricted bicyclic peptide technology. Nectin-4 is a cell adhesion protein overexpressed in various human cancers that can benefit from CD137 agonist activity. BT7480 is a novel, first-in-class Nectin-4 / CD137 tumor-targeting immune cell agonist (TICA).

[0070] It is well known that several major solid tumor types express Nectin-4 and can infiltrate immune cells to varying degrees. Compounds that activate CD137 only when co-linked with Nectin-4 require access from relevant immune tumor cells. Analysis of RNA expression data from human tumor samples within the Cancer Genome Atlas (TCGA) indicates that Nectin-4 and CD137 are co-expressed in several tumor types, including lung, breast, esophagus, stomach, ovary, head and neck, pancreas, and bladder (see [link to TCM description]). Figure 10A More than half of the tumor types examined had a substantial proportion of tumors expressing high levels of Nectin-4 and CD137 (see [link]). Figure 10ABased on these data, three major tumor types, namely NSCLC, HNSCC, and bladder cancer, were selected to further examine Nectin-4 and CD137 in human tumors through spatial proteomic distribution and image analysis (see Example 3).

[0071] The level of Nectin-4 protein in tumor tissue has been measured using multiplex immunofluorescence (mIF) assays. The levels of Nectin-4 protein on the tumor cell membrane and in the cytoplasm of tumor cells have been found to indicate a response to treatment with a bicyclic tumor-targeting immunoagonist (TICA) specific to Nectin-4. Without being bound by any particular theory or mechanism, the inventors have found that tumors with elevated Nectin-4 protein levels in diseased tissue are more likely to benefit from treatment with a bicyclic tumor-targeting immunoagonist (TICA) specific to Nectin-4. It has also been found that tumors with elevated Nectin-4 protein levels on the tumor cell membrane are more likely to benefit from treatment with BT7480.

[0072] Therefore, in one aspect, the present invention provides a method for identifying or selecting patients with elevated levels of Nectin-4 protein and / or RNA expression in diseased tissue, the method comprising measuring the expression levels of Nectin-4 protein and / or RNA in the diseased tissue of the patient, and selecting patients with elevated levels of Nectin-4 protein and / or RNA expression in the diseased tissue.

[0073] In another aspect, this article provides a method for treating a patient with elevated levels of Nectin-4 protein and / or RNA expression in diseased tissue (e.g., as determined using the methods described herein), the method comprising administering to the patient a bicyclic tumor-targeting immune agonist (TICA) specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0074] In another aspect, the present invention provides a method for treating a patient’s disease, the method comprising selecting a patient with elevated levels of Nectin-4 protein and / or RNA expression in diseased tissue, for example, using the method described herein, and administering to the patient a bicyclic tumor-targeting immune agonist (TICA) specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0075] 2. Compounds and definitions:

[0076] As used herein, the term "bicyclic toxin conjugate specific to Nectin-4" refers to a bicyclic toxin conjugate that specifically binds to Nectin-4. Several bicyclic toxin conjugates specific to Nectin-4 have previously been described, for example, in US 2019 / 03889906, WO 2019 / 243832, and WO 2019 / 243833, the contents of which are incorporated herein by reference in their entirety. In US 2019 / 03889906, WO 2019 / 243832, and WO 2019 / 243833, BT8009 is referred to as BCY8245.

[0077] As used herein, the term "BT8009" is a bicyclic toxin conjugate or a pharmaceutically acceptable salt thereof having the structure shown below, wherein the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tripropyl-2-en-1-one (TATA), and the peptide ligand comprises the following amino acid sequence:

[0078] (β-Ala)-Sar 10 -C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1)

[0079] Where Sar represents sarcosine, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, and C i C ii and C iii This indicates the first, second, and third cysteine ​​residues.

[0080]

[0081]

[0082] As used herein, the term "bicyclic tumor-targeting immune agonist (TICA) specific to Nectin-4" refers to a bicyclic tumor-targeting immune agonist (TICA) that specifically binds to Nectin-4. Various bicyclic tumor-targeting immune agonists (TICAs) specific to Nectin-4 have previously been described, for example, in US 2019 / 0307836, WO 2019 / 193328, US 2021 / 0040154, WO 2021 / 019244, and WO 2021 / 019246 (the contents of which are incorporated herein by reference in their entirety). In US 2021 / 0040154, WO 2021 / 019244, and WO 2021 / 019246, BT7480 is referred to as BCY11863. The term "BT7480" is a bicyclic tumor-targeting immune agonist (TICA), which is a tandem heterobicyclic peptide complex consisting of two CD137-specific peptides linked by an N-(acid-PEG3)-N-bis(PEG3-azide) linker, having the structure shown below.

[0083]

[0084] As used herein, the term "pharmaceutically acceptable salt" means those salts that, within reasonable medical judgment, are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. described 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 with an amino group formed with an inorganic or organic acid, or by other methods used in the art, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid; organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid; and other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glyceryl phosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, trimethylacetate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0085] Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed when appropriate using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate ions. It should be understood that salt forms are within the scope of this invention, and references to peptide ligands include salt forms of said ligands.

[0086] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Typically, such salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water, in an organic solvent, or in a mixture of both.

[0087] Unless otherwise stated, the structures described herein are also intended to include all isomers (e.g., enantiomers, diastereomers, and geometric (or conformations)) of such structures; for example, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, individual stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric (or conformations), are all within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention. Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, those having hydrogen replaced by deuterium or tritium, or those enriched with... 13 C or 14 Compounds with this structure in which carbon replaces carbon (C) are all within the scope of this invention. Such compounds can be used, for example, as analytical tools, probes in bioassays, or therapeutic agents according to the invention.

[0088] As used herein, the term “about” or “approximately” has the meaning of 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.

[0089] 3. Description of exemplary embodiments of the present invention

[0090] In one aspect, the present invention provides a method for identifying or selecting patients with elevated Nectin-4 protein levels in diseased tissue, the method comprising measuring Nectin-4 protein levels in the patient's diseased tissue and selecting patients with elevated Nectin-4 protein levels in their diseased tissue. Nectin-4 protein levels in diseased tissue can be measured using various methods.

[0091] 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 the staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, and selecting patients who are positive for staining in the Nectin-4 IHC staining assay.

[0092] As used herein, the term "stain-positive patient" refers to a patient who has a certain percentage of stain-positive cells in a tumor tissue section as determined by the Nectin-4 IHC staining assay. In some embodiments, a stain-positive patient has approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of stain-positive cells in a tumor tissue section as determined by the Nectin-4 IHC staining assay.

[0093] There are various methods for measuring staining intensity in IHC staining assays. In some embodiments, staining intensity is measured by visual scoring, such as manual scoring using a conventional optical microscope. In some embodiments, staining intensity is measured by calculating a tissue analysis (CTA) score. Staining intensity levels can be no staining (0), weak staining (1+), median staining (2+), or strong staining (3+). In some embodiments, staining intensity is measured on the tumor cell membrane of a tumor tissue section. In some embodiments, staining intensity is measured in the cytoplasm of tumor cells in a tumor tissue section. In some embodiments, staining intensity is measured both on the tumor cell membrane and in the cytoplasm of tumor cells in a tumor tissue section.

[0094] In some embodiments, a positive staining result means an H score of about 15 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 20 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 30 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 40 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 50 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 75 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 100 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 125 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of about 150 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 200 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 300 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 400 or higher in a tumor tissue section using an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 500 or higher in a tumor tissue section using an IHC staining assay.

[0095] The H score is the sum of the percentage of cells multiplied by the staining intensity of their respective grades from 0 to 3 (no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+)) as described above.

[0096] [((0×(% of cells at 0))+((1×(% of cells at 1+))+((2×(% of cells at 2+))+((3×(% of cells at 3))]

[0097] H-scores can be generated for different compartments (including, for example, tumor cell membranes and cytoplasm) in tumor tissue sections. In some embodiments, the H-score refers to the H-score of the tumor cell membrane, which is the sum of the products of cell percentage and their cell membrane staining intensities according to a 0-3 scale as described above. In some embodiments, the H-score refers to the H-score of the tumor cell cytoplasm, which is the sum of the products of cell percentage and their cytoplasmic staining intensities according to a 0-3 scale as described above.

[0098] In some embodiments, a positive staining result means an H score of about 15 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 20 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 30 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 40 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 50 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 75 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of about 100 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 125 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 150 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 200 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 300 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 400 or higher for the tumor cell membrane in an IHC staining assay. In some embodiments, a positive staining result means an H score of approximately 500 or higher for the tumor cell membrane in an IHC staining assay.

[0099] In some embodiments, a positive staining result means an H score of about 15 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 20 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 30 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 40 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 50 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 75 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means an H score of about 100 or higher for the cytoplasm of tumor cells in a tumor tissue section according to an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 125 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 150 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 200 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 300 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 400 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the H score of the cytoplasm of tumor cells in a tumor tissue section is about 500 or higher in an IHC staining assay.

[0100] In some embodiments, a positive staining result means that the combined H score of the tumor cell membrane and tumor cell cytoplasm in a tumor tissue section is about 15 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 20 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 30 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 40 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 50 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 75 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the tumor cell cytoplasm in a tumor tissue section is about 100 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 125 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 150 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 200 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 300 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 400 or higher in an IHC staining assay. In some embodiments, a positive staining result means that the combined H score of the cytoplasm of tumor cells in a tumor tissue section is about 500 or higher in an IHC staining assay.

[0101] 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 the 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 higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher.

[0102] 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 the 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 higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher.

[0103] 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 the staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, and selecting patients whose tumor cells have an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher.

[0104] In some embodiments, the present invention provides a method for treating cancer in a patient with elevated Nectin-4 protein levels in tumor tissue, the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0105] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with elevated Nectin-4 protein levels in tumor tissue, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0106] In some embodiments, the present invention provides a method for treating a patient's cancer, the method comprising measuring the level of Nectin-4 protein in a section of the patient's tumor tissue, selecting patients with elevated Nectin-4 protein levels in their tumor tissue, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0107] In some implementations, the cancer is pancreatic cancer. In some implementations, the cancer is stomach cancer. In some implementations, the cancer is bladder cancer. In some implementations, the cancer is head and neck cancer. In some implementations, the cancer is non-small cell lung cancer (NSCLC). In some implementations, the cancer is triple-negative breast cancer (TNBC). In some implementations, the cancer is ovarian cancer.

[0108] In some embodiments, the present invention provides a method for treating cancer in patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher in tumor tissue sections according to a Nectin-4 IHC staining assay, said method comprising administering to the patient a bicyclic toxin conjugate specific to 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.

[0109] In some embodiments, the present invention provides a method for treating cancer in patients whose tumor cell membrane H score in tumor tissue sections is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, according to a Nectin-4 IHC staining assay, the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0110] In some embodiments, the present invention provides a method for treating cancer in patients whose cytoplasmic H score of tumor cells in tumor tissue sections is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, according to a Nectin-4 IHC staining assay, the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0111] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher in tumor tissue sections as determined by Nectin-4 staining, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0112] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient whose tumor cell membrane H score in a tumor tissue section is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0113] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient whose H score of the cytoplasm of tumor cells in a tumor tissue section is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0114] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising measuring the staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0115] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising measuring the staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0116] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising measuring the staining intensity in tumor tissue sections of the patient using a Nectin-4 IHC staining assay, selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, about 150 or higher, about 200 or higher, about 300 or higher, about 400 or higher, or about 500 or higher, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0117] In some implementations, the level of Nectin-4 protein in diseased tissue can be measured by measuring the level of Nectin-4 mRNA in the diseased tissue.

[0118] In some embodiments, clinically common tests are used to measure Nectin-4 protein levels in diseased tissue. In some embodiments, Nectin-4 protein levels in diseased tissue are measured by measuring the DNA copy number of other biomarkers as described herein, such as Nectin-4, SDHC, and / or DDR2. In some embodiments, Nectin-4 protein levels in diseased tissue can be measured by measuring the Nectin-4 DNA copy number in the diseased tissue. In some embodiments, Nectin-4 protein levels in diseased tissue can be measured by measuring the SDHC DNA copy number in the diseased tissue. In some embodiments, Nectin-4 protein levels in diseased tissue can be measured by measuring the DDR2 DNA copy number in the diseased tissue.

[0119] 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 the copy number of Nectin-4 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting patients with elevated Nectin-4 DNA copy numbers in tumor tissue. 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 the copy number of SDHC DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting patients with elevated SDHC DNA copy numbers in tumor tissue. 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 the copy number of DDR2 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA), and selecting patients with elevated DDR2 DNA copy numbers in tumor tissue. In some embodiments, the method further comprises administering to the patient with elevated Nectin-4 protein levels in tumor tissue a bicyclic toxin conjugate specific to Nectin-4 (e.g., BT8009) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0120] In some implementations, the patient is a patient with pancreatic cancer. In some implementations, the patient is a patient with stomach cancer. In some implementations, the patient is a patient with bladder cancer. In some implementations, the patient is a patient with head and neck cancer. In some implementations, the patient is a patient with non-small cell lung cancer (NSCLC). In some implementations, the patient is a patient with triple-negative breast cancer (TNBC). In some implementations, the patient is a patient with ovarian cancer.

[0121] 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 stomach / upper gastrointestinal (GI) tumor tissue. In some embodiments, the tumor tissue is pancreatic tumor tissue. In some embodiments, the tumor tissue is urinary tract epithelial 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.

[0122] As used herein, the terms “increase,” “elevation,” or “enhancement” are used interchangeably and cover any measurable increase in biological function and / or biological activity and / or concentration. For example, an increase can be an increase of 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 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 25 times, about 50 times, about 100 times, or more, relative to a control or baseline amount of function or activity or concentration.

[0123] As used herein, the term "substance in sample" refers to substances in the sample (e.g., Nectin-4 protein, Nectin-4 mRNA, Nectin-4 DNA copy number, SDHC DNA copy number, or DDR2). The “elevated level” of the substance (DNA copy number) means that the amount of the substance is increased by 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 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 25 times, about 50 times, about 100 times or more, as determined by techniques known in the art, relative to the amount of the substance in one or more control samples (such as individuals without disease or condition (e.g., cancer) or a group of individuals or internal controls). An subject may also be identified as having an “elevated level” of a substance if the concentration of the substance increases by one, two, three, four, five, or more standard deviations relative to the mean (mean) or median of the substance in a retrospective analysis of samples from the control group, baseline group, or patient samples. Such control or baseline levels may be predetermined, measured prior to measurement in the sample, or available from a database of such control samples, as is practiced in the art. In other words, control and subject samples do not necessarily need to 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.

[0124] As used herein, the term "Nectin-4 amplification" refers to an increase in the Nectin-4 DNA copy number relative to normal tissue. In some embodiments, this is expressed as the copy number ratio Log2 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 implementations, the Nectin-4 Log2 (CN ratio) is >1.0.

[0125] As used herein, the term "SDHC amplification" refers to an increase in the copy number of SDHC DNA relative to normal tissue. In some embodiments, this is expressed as the copy number ratio Log2 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.

[0126] As used herein, the term "DDR2 amplification" refers to an increase in the DDR2 DNA copy number relative to normal tissue. In some embodiments, this is expressed as the copy number ratio Log2 or 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.

[0127] There are several methods for measuring the DNA copy number of Nectin-4, SDHC, and DDR2 in tissues. In some implementations, methods for measuring the DNA copy number of Nectin-4, SDHC, or DDR2 in a patient's tumor tissue or circulating tumor DNA (ctDNA) include whole-exome sequencing.

[0128] 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 Nectin-4 DNA copy number and selecting patients with elevated Nectin-4 DNA copy number.

[0129] 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 SDHC DNA copy number and selecting patients with elevated SDHC DNA copy number.

[0130] 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 DDR2 DNA copy number and selecting patients with elevated DDR2 DNA copy number.

[0131] 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 the Nectin-4 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) slices using whole-exome sequencing, and selecting patients with a Nectin-4 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.

[0132] 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 the SDHC DNA copy number in the patient’s tumor tissue or circulating tumor DNA (ctDNA) slices using whole-exome sequencing, and selecting patients with 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.

[0133] 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 the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) slices using whole-exome sequencing, and selecting patients with a DDR2 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.

[0134] In some embodiments, the present invention provides a method for treating cancer in 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 methods as described herein), the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0135] In some embodiments, the present invention provides a method for treating a patient with cancer, the method 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 methods as described herein), and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0136] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising measuring the level of Nectin-4 protein in a section of the patient's tumor tissue, selecting a patient with elevated Nectin-4 protein levels in the tumor tissue (e.g., identified as having elevated Nectin-4 protein levels in diseased tissue using the methods described herein), and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the elevated Nectin-4 protein level is as described herein.

[0137] In some implementations, the cancer is pancreatic cancer. In some implementations, the cancer is stomach cancer. In some implementations, the cancer is bladder cancer. In some implementations, the cancer is head and neck cancer. In some implementations, the cancer is non-small cell lung cancer (NSCLC). In some implementations, the cancer is triple-negative breast cancer (TNBC). In some implementations, the cancer is ovarian cancer.

[0138] In some embodiments, the bicyclic toxin conjugates specific to Nectin-4 are selected from compounds described in US 2019 / 03889906, WO 2019 / 243832 and WO 2019 / 243833, each of which is incorporated herein by reference in its entirety.

[0139] In some implementations, the bicyclic toxin conjugate specific to Nectin-4 is BT8009 as described herein or a pharmaceutically acceptable salt thereof.

[0140] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein in tumor tissue, the method comprising administering BT8009 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0141] In some embodiments, the present invention provides a method for treating a patient’s cancer, the method comprising selecting a patient with 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.

[0142] In some embodiments, the present invention provides a method for treating a patient’s cancer, the method comprising measuring the level of Nectin-4 protein in the patient’s tumor tissue, selecting patients with elevated levels of Nectin-4 protein in their tumor tissue, and administering BT8009 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0143] Bicyclic toxin conjugates specific to Nectin-4, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, may be administered to patients in a variety of dosage ranges.

[0144] In some embodiments, the method of the present invention includes administering to a patient a bicyclic toxin conjugate specific to Nectin-4 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 method of the present invention includes administering to a patient a bicyclic toxin conjugate specific to Nectin-4 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.

[0145] In some implementation schemes, at approximately 1-27 mg / m² 2 The pharmaceutical composition of the present invention is administered at a dosage of about 2-20 mg / m³. In some embodiments, the dosage is about 2-20 mg / m³. 2 The pharmaceutical composition of the present invention is administered at a dosage of about 2-20 mg / m³. In some embodiments, the dosage is about 2-20 mg / m³. 2 The pharmaceutical composition of the present invention is administered at a dosage of approximately 2.2, 4.4, 7.3, 11, 14.6, or 19.4 mg / m³. In some embodiments, the dosage is approximately 2.2, 4.4, 7.3, 11, 14.6, or 19.4 mg / m³. 2 The pharmaceutical composition of the present invention is administered at a dosage of about 2.5, 5.0, 7.5, 10.0, 13.0, or 17.0 mg / m³. In some embodiments, the dosage is about 2.5, 5.0, 7.5, 10.0, 13.0, or 17.0 mg / m³. 2 The pharmaceutical composition of the present invention is administered at a dosage of approximately 1.5-3.5, 3.5-5.5, 6.5-8.5, 10-12, 13.5-15.5, or 18.5-20.5 mg / m³. 2 The pharmaceutical composition of the present invention is administered at a dosage of about 1-10 or 10-20 mg / m³. 2The pharmaceutical composition of the present invention is administered at a dosage of about 21, 22, 23, 24, 25, 26, or 27 mg / m³. In some embodiments, the dosage is about 21, 22, 23, 24, 25, 26, or 27 mg / m³. 2 The pharmaceutical composition of the present invention is administered at the prescribed dosage.

[0146] Bicyclic toxin conjugates specific to Nectin-4, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, can be administered to patients at various dosage frequencies. In some embodiments, the method of the present invention includes administering to a patient a bicyclic toxin conjugate specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dosage frequency of once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days. In some embodiments, the method of the present invention includes administering to a patient a bicyclic toxin conjugate specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, at a dosage frequency of twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks.

[0147] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue, the method 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 levels of Nectin-4 protein and / or RNA expression are as described herein.

[0148] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with elevated levels of Nectin-4 protein and / or RNA expression 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 levels of Nectin-4 protein and / or RNA expression are as described herein.

[0149] In some embodiments, the present invention provides a method for identifying or selecting patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue, the method comprising measuring the expression levels of Nectin-4 protein and / or RNA in the patient's tumor tissue, and selecting patients with elevated levels of Nectin-4 protein and / or RNA expression in their tumor tissue. In some embodiments, the method further comprises administering to the patient with elevated levels of Nectin-4 protein and / or RNA expression in their tumor tissue a bicyclic TICA specific for Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0150] In some implementations, the patient is a patient with pancreatic cancer. In some implementations, the patient is a patient with stomach cancer. In some implementations, the patient is a patient with bladder cancer. In some implementations, the patient is a patient with head and neck cancer. In some implementations, the patient is a patient with non-small cell lung cancer (NSCLC). In some implementations, the patient is a patient with triple-negative breast cancer (TNBC). In some implementations, the patient is a patient with ovarian cancer.

[0151] 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.

[0152] As used herein, the term "elevated Nectin-4 protein level" refers to a certain percentage of cells in tumor tissue having a detectable amount 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 means that approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of cells in tumor tissue have a detectable amount of Nectin-4 protein, for example, on the tumor cell membrane or in the tumor cell cytoplasm, or both.

[0153] There are several methods for measuring the amount of Nectin-4 protein in tissues. In some embodiments, methods for measuring Nectin-4 protein levels in patient tumor tissue include using a Nectin-4 multiplex immunofluorescence (mIF) assay. In some embodiments, the Nectin-4 mIF assay includes staining tumor tissue sections with 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 that selectively binds to the ECD of Nectin-4 is the rabbit monoclonal α-Nectin-4 primary antibody YMW-1-58.

[0154] In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is up to about 50 μg / mL. In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is up to about 40 μg / mL. In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is up to about 30 μg / mL. In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is up to about 20 μg / mL. In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is up to about 10 μg / mL. In some embodiments, the concentration of rabbit monoclonal α-Nectin-4 primary antibody is 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 concentration of rabbit monoclonal α-Nectin-4 primary antibody (such as YMW-1-58) selectively binding to the ECD of Nectin-4 is 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 concentration of rabbit monoclonal α-Nectin-4 primary antibody (such as YMW-1-58) selectively binding to the ECD of Nectin-4 is about 10 μg / mL.

[0155] 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 mIF assay, and selecting patients who are positive for staining in the Nectin-4 mIF assay. In some embodiments, the Nectin-4 mIF assay is as described in Example 3 herein.

[0156] As used herein, the term "stain-positive patient" refers to a patient who has a certain percentage of stain-positive cells in a tumor tissue section as determined by the Nectin-4mIF assay. In some embodiments, a stain-positive patient has approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% of stain-positive cells in a tumor tissue section as determined by the Nectin-4mIF assay.

[0157] There are various methods for measuring staining intensity in mIF assays. In some embodiments, staining intensity is measured by visual scoring, such as manual scoring using a conventional optical microscope. In some embodiments, staining intensity is measured by calculating a tissue analysis (CTA) score. Staining intensity levels can be no staining (0), weak staining (1+), median staining (2+), or strong staining (3+). In some embodiments, staining intensity is measured on the tumor cell membrane of a tumor tissue section. In some embodiments, staining intensity is measured in the cytoplasm of tumor cells in a tumor tissue section. In some embodiments, staining intensity is measured both on the tumor cell membrane and in the cytoplasm of tumor cells in a tumor tissue section.

[0158] In some embodiments, staining positivity refers to an H score of about 15 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 20 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 30 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 40 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 50 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 75 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 100 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 125 or higher in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H score of about 150 or higher in a tumor tissue section as determined by the mIF assay.

[0159] The H score is the sum of the percentage of cells multiplied by the staining intensity of their respective grades from 0 to 3 (no staining (0), weak staining (1+), moderate staining (2+), or strong staining (3+)) as described above.

[0160] [((0×(% of cells at 0))+((1×(% of cells at 1+))+((2×(% of cells at 2+))+((3×(% of cells at 3))]

[0161] H-scores can be generated for different compartments (including, for example, tumor cell membranes and cytoplasm) in tumor tissue sections. In some embodiments, the H-score refers to the H-score of the tumor cell membrane, which is the sum of the products of cell percentage and their cell membrane staining intensities according to a 0-3 scale as described above. In some embodiments, the H-score refers to the H-score of the tumor cell cytoplasm, which is the sum of the products of cell percentage and their cytoplasmic staining intensities according to a 0-3 scale as described above.

[0162] In some embodiments, staining positivity refers to an H-score of about 15 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 20 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 30 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 40 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 50 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 75 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 100 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some embodiments, staining positivity refers to an H-score of about 125 or higher for the tumor cell membrane in a tumor tissue section as determined by the mIF assay. In some implementations, a positive staining result means that the H score of the tumor cell membrane in a tumor tissue section is approximately 150 or higher in the mIF assay.

[0163] In some embodiments, staining positivity refers to an H score of about 15 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 20 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 30 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 40 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 50 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 75 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of about 100 or higher for the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of approximately 125 or higher in the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay. In some embodiments, staining positivity refers to an H score of approximately 150 or higher in the cytoplasm of tumor cells in a tumor tissue section as determined by mIF assay.

[0164] 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 mIF assay, and selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher.

[0165] 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 mIF assay, and selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher.

[0166] 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 mIF assay, and selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher.

[0167] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue, the method 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 levels of Nectin-4 protein and / or RNA expression are as described herein.

[0168] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with elevated levels of Nectin-4 protein and / or RNA expression 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 levels of Nectin-4 protein and / or RNA expression are as described herein.

[0169] In some embodiments, the present invention provides a method for treating a patient's cancer, the method comprising measuring the expression levels of Nectin-4 protein and / or RNA in tumor tissue sections of the patient, selecting patients with elevated Nectin-4 protein and / or RNA expression levels in their tumor tissues, 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.

[0170] In some implementations, the cancer is pancreatic cancer. In some implementations, the cancer is stomach cancer. In some implementations, the cancer is bladder cancer. In some implementations, the cancer is head and neck cancer. In some implementations, the cancer is non-small cell lung cancer (NSCLC). In some implementations, the cancer is triple-negative breast cancer (TNBC). In some implementations, the cancer is ovarian cancer.

[0171] In some embodiments, the present invention provides a method for treating cancer in patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in tumor tissue sections according to a Nectin-4 mIF assay, said method comprising administering to the patient a bicyclic TICA specific to 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.

[0172] In some embodiments, the present invention provides a method for treating cancer in patients whose tumor cell membrane H score in tumor tissue sections is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in a Nectin-4 mIF assay, the method comprising administering to the patient a bicyclic TICA specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0173] In some embodiments, the present invention provides a method for treating cancer in patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in tumor cell cytoplasm in tumor tissue sections as determined by Nectin-4 mIF assay, said method comprising administering to the patient a bicyclic TICA specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0174] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in a tumor tissue section as determined by a Nectin-4 mIF assay, and administering to the patient a bicyclic TICA specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0175] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient whose H score of tumor cell membrane in a tumor tissue section is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in a Nectin-4 mIF assay, and administering to the patient a bicyclic TICA specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0176] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient whose H score in the cytoplasm of tumor cells in a tumor tissue section is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in a Nectin-4 mIF assay, and administering to the patient a bicyclic TICA specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.

[0177] In some embodiments, the present invention provides a method for treating a patient with cancer, the method 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 higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher, and administering to the patient a bicyclic TICA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof that is specific to Nectin-4.

[0178] In some embodiments, the present invention provides a method for treating a patient with cancer, the method 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 higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher, and administering to the patient a bicyclic TICA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof that is specific to Nectin-4.

[0179] In some embodiments, the present invention provides a method for treating a patient with cancer, the method 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 higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher, and administering to the patient a bicyclic TICA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof that is specific to Nectin-4.

[0180] In some implementations, the bicyclic TICA that is specific to Nectin-4 is BT7480 as described herein or a pharmaceutically acceptable salt thereof.

[0181] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue, the method comprising administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0182] In some embodiments, the present invention provides a method for treating cancer in patients whose tumor cell membrane H score in tumor tissue sections is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher in a Nectin-4mIF assay, the method comprising administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0183] In some embodiments, the present invention provides a method for treating a patient’s cancer, the method comprising selecting a patient with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue, and administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0184] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient whose tumor cell membrane H score in a tumor tissue section as determined by mIF assay is about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher, and administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0185] In some embodiments, the present invention provides a method for treating a patient’s cancer, the method comprising measuring the expression levels of Nectin-4 protein and / or RNA in the patient’s tumor tissue, selecting patients with elevated levels of Nectin-4 protein and / or RNA in their tumor tissue, and administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0186] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising measuring staining intensity in tumor tissue sections of the patient using a Nectin-4mIF assay, selecting patients with an H score of about 15 or higher, about 20 or higher, about 30 or higher, about 40 or higher, about 50 or higher, about 75 or higher, about 100 or higher, about 125 or higher, or about 150 or higher, and administering BT7480 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof to the patient.

[0187] Bicyclic TICA, which is specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be administered to patients in a variety of dose ranges.

[0188] In some embodiments, the method of the present invention includes administering to a patient a bicyclic TICA specific to Nectin-4, 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 method of the present invention includes administering to a patient a bicyclic TICA specific to Nectin-4, 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.

[0189] In some embodiments, the method of the present invention includes using approximately 100 mg / m³ 2 Or a smaller dose may be administered 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 method of the present invention comprises administering approximately 90 mg / m²... 2 Approximately 80 mg / m 2 Approximately 70 mg / m 2 Approximately 60 mg / m 2 Approximately 50 mg / m 2 Approximately 40 mg / m 2 Approximately 30 mg / m 2 Approximately 25 mg / m 2 Approximately 22.5 mg / m 2 Approximately 20 mg / m2 Approximately 17.5 mg / m³ 2 Approximately 15 mg / m 2 Approximately 12.5 mg / m 2 Approximately 10 mg / m 2 Approximately 7.5 mg / m 2 Approximately 5mg / m 2 Approximately 2.5 mg / m 2 or about 1 mg / m 2 The method of the present invention involves administering to the 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 / m². 2 Approximately 25 mg / m 2 The patient is given a dose of bicyclic TICA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof that is specific to Nectin-4.

[0190] Bicyclic TICA specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be administered to a patient at various dosage frequencies. In some embodiments, the method of the present invention includes administering a bicyclic TICA specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient at a dosage frequency of once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days. In some embodiments, the method of the present invention includes administering a bicyclic TICA specific to Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient at a dosage frequency of twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks.

[0191] 4. Preparation and application

[0192] In some embodiments, the method described herein includes administering a pharmaceutical composition comprising a bicyclic toxin conjugate specific to Nectin-4 as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or mediator. In some embodiments, the bicyclic toxin conjugate specific to Nectin-4, or a pharmaceutically acceptable salt thereof, is formulated for intravenous administration to a patient.

[0193] In some embodiments, the method described herein includes administering a pharmaceutical composition comprising a bicyclic TICA specific for Nectin-4 as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or mediator. In some embodiments, a bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, is formulated for intravenous administration to a patient.

[0194] The term "pharmaceutically acceptable carrier, adjuvant, or mediator" refers to a non-toxic carrier, adjuvant, or mediator that does not impair the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable carriers, adjuvants, or mediators that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0195] The compositions of the present invention can be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, orally, via an implanted reservoir. As used herein, the term “parenterally” includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheathic, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of the present invention can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable formulation can 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. Acceptable media and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are commonly used as solvents or suspension media.

[0196] To achieve this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glycerol derivatives are suitable for the preparation of injectable formulations, as are natural, pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylene form. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other commonly used surfactants, such as Tween and Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used to achieve the formulation purpose.

[0197] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspending agents are to be taken orally, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may also be added.

[0198] Alternatively, the pharmaceutically acceptable compositions of the present invention can be used for administration in the form of rectal suppositories. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0199] The pharmaceutically acceptable compositions of the present invention can also be applied topically, particularly when the therapeutic target includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower intestine. Suitable topical formulations are readily prepared for each of these areas or organs.

[0200] Local application to the lower intestine can be achieved with rectal suppositories (see above) or with a suitable enema. Topical transdermal patches may also be used.

[0201] For topical application, the provided pharmaceutically acceptable compositions can be formulated into suitable ointments containing active ingredients suspended or dissolved in one or more carriers. Carriers for topical application of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the provided pharmaceutically acceptable compositions can be formulated into suitable lotions or creams containing active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, dehydrated sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

[0202] For ocular use, the provided pharmaceutically acceptable composition may be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline, with or without a preservative, such as benzalkonium chloride. Alternatively, for ocular use, the pharmaceutically acceptable composition may be formulated as an ointment such as petrolatum.

[0203] The pharmaceutically acceptable compositions of the present invention can also be administered via nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the field of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, bioavailability enhancers, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0204] Most preferably, the pharmaceutically acceptable composition of the present invention is formulated for oral administration. Such a formulation may or may not be administered with food. In some embodiments, the pharmaceutically acceptable composition of the present invention is administered without food. In other embodiments, the pharmaceutically acceptable composition of the present invention is administered with food.

[0205] The amount of the compounds of the present invention, which can be combined with carrier materials to produce compositions of a single dosage form, will vary depending on the host being treated and the specific mode of administration. Preferably, the provided compositions should be formulated to a dose of 0.01-1 mg / kg body weight / day for administration to patients receiving these compositions.

[0206] 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 compound used, age, weight, general health condition, sex, diet, timing of administration, excretion rate, drug combination, the judgment of the attending physician, and the severity of the specific disease being treated. The amount of the compound of the present invention in the composition will also depend on the specific compound in the composition.

[0207] 5. Applications

[0208] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with elevated levels of Nectin-4 protein in tumor tissue, for example, using the method described herein, and administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the treatment further includes, for example, measuring the copy number of Nectin-4 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA) using whole-exome sequencing as described herein.

[0209] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein in tumor tissue, the method comprising administering to the patient a bicyclic toxin conjugate specific to Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof. In some embodiments, the treatment also includes, for example, measuring the copy number of Nectin-4 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA) using whole-exome sequencing as described herein.

[0210] In some embodiments, the present invention provides a method for treating a patient with cancer, the method comprising selecting a patient with elevated levels of Nectin-4 protein in tumor tissue, for example, using the method 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 also includes, for example, measuring the copy number of Nectin-4 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA) using whole-exome sequencing as described herein.

[0211] In some embodiments, the present invention provides a method for treating cancer in patients with elevated levels of Nectin-4 protein in tumor tissue, the method 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 also includes, for example, measuring the copy number of Nectin-4 DNA in the patient's tumor tissue or circulating tumor DNA (ctDNA) using whole-exome sequencing as described herein.

[0212] smallpox

[0213] The cancers or proliferative disorders or tumors treated using the methods and uses described herein include, but are not limited to, blood cancers, lymphoma, myeloma, leukemia, neurocancer, skin cancer, breast cancer, prostate cancer, colorectal cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, kidney cancer, and vascular cancer.

[0214] The cancers treated using the methods described herein can be selected from colorectal cancers, such as microsatellite stable (MSS) metastatic colorectal cancer, including advanced or progressive microsatellite stable (MSS) CRC; non-small cell lung cancer (NSCLC), such as 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 implementations, the cancer is cervical cancer. In some implementations, the cancer is head and neck cancer. In some implementations, the cancer is stomach cancer. In some implementations, the cancer is gastroesophageal junction cancer. In some implementations, the cancer is bladder cancer.

[0215] In some implementations, cancer includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenström macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, 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, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0216] In some implementations, the cancer is glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0217] In some implementations, the cancer is an 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, brainstem glioma, ependymoma, mixed glioma, optic glioma, subependymal tumor, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or schwannoma. In some implementations, the cancer is a type more common in children than in adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some implementations, the patient is an adult. In some implementation schemes, the patient is a child or a pediatric patient.

[0218] In another implementation, the cancers include, but are not limited to, mesothelioma, hepatobiliary duct (liver and bile duct), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (stomach cancer, colorectal cancer, and duodenal cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, and kidney cancer. Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, non-Hodgkin's lymphoma, spinal tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, bile duct cancer, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the aforementioned cancers.

[0219] In some implementations, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UP SC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary duct cancer; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; undifferentiated thyroid carcinoma; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0220] In some implementation schemes, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0221] In some implementations, the cancer is a solid tumor, such as sarcoma, carcinoma, or lymphoma. Solid tumors typically comprise abnormal masses of tissue that do not usually include cysts or fluid-filled areas. In some implementations, the cancer is selected from renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal cancer 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 cancer, ovarian tumor, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer. Hepatobiliary duct carcinoma; soft tissue and synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; undifferentiated thyroid carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland carcinoma; glioma or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0222] In some implementation schemes, the cancer is selected from renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial cancer, ovarian tumor, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, undifferentiated thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, brain cancer, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0223] In some implementation schemes, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian tumor, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, undifferentiated thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0224] 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 an ovarian tumor. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is papillary serous uterine carcinoma (UPSC). In some embodiments, the cancer is hepatobiliary duct cancer. 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 undifferentiated thyroid 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 schwannoma (MPNST). In some embodiments, the cancer is neurofibromatosis-1-associated MPNST. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0225] In some implementation schemes, cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, atypical teratoid tumor / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors, astrocytomas, brain and spinal cord tumors, brainstem gliomas, atypical teratoid tumors / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, cancers of unknown primary origin, central nervous system cancers, cervical cancer, childhood cancers, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, and craniopharyngioma. Cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing sarcoma, extracranial germ cell tumors, gonadal germ cell tumors, extrahepatic bile duct cancer, ocular cancer, osteofibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, histiocytosis, Langerhans cell carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip cancer. Cancer and oral cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, AIDS-related lymphoma, macroglobulinemia, male breast cancer, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, malignant mesothelioma, occult primary metastatic squamous neck cancer, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine adenoma syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloma, multiple myeloma, chronic myeloproliferative disorder, nasal cavity cancer, sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer. Cancer, oral cancer, lip cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, sinus cancer, nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated solid tumors of the pineal gland, pineal blastoma, pituitary adenoma, plasmacytoma, pleural pulmonary blastoma, breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, clear cell renal cell carcinoma, renal pelvis cancer, ureteral cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Cezari syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, occult primary squamous neck cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer,Supratentorial primitive neuroectodermal tumors, T-cell lymphomas, testicular cancer, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, triple-negative breast cancer (TNBC), gestational trophoblastic tumors, unknown primary cancers, childhood abnormal cancers, urethral cancer, uterine cancer, uterine sarcoma, Waldenström macroglobulinemia, or Wilms' tumor.

[0226] In some implementations, 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 cancer, and squamous cell carcinoma), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), melanoma, multiple myeloma (MM), ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer (including clear cell renal carcinoma and papillary renal carcinoma), and gastric cancer.

[0227] In some implementation schemes, 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 blood cancers.

[0228] In some implementations, the cancer is small cell lung cancer, non-small cell lung cancer, colorectal cancer, multiple myeloma, or AML.

[0229] The invention is further characterized by methods and compositions for diagnosing, prognosing, and treating virus-associated cancers, including human immunodeficiency virus (HIV)-associated solid tumors, human papillomavirus (HPV)-16-positive incurable solid tumors, and adult T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by a highly aggressive form of CD4+ T-cell leukemia with clonal integration of HTLV-I into leukemic cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); as well as virus-associated tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma. (See https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; also see https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892)

[0230] In some embodiments, cancer or tumor includes any cancer described herein. In some embodiments, cancer includes melanoma. In some embodiments, cancer includes breast cancer. In some embodiments, cancer includes lung cancer. In some embodiments, cancer includes small cell lung cancer (SCLC). In some embodiments, cancer includes non-small cell lung cancer (NSCLC).

[0231] 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% relative 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% relative to the amount of cancer or tumor before treatment.

[0232] According to the method of the invention, compounds and compositions can be administered in any amount and via any route of administration that is effective in treating cancer or tumors or reducing their severity. The exact amount required varies from subject to subject, depending on the subject's species, age and general condition, severity of disease or symptom, specific agent, mode of administration, etc. According to the method of the invention, compounds and compositions are preferably formulated in a dosing unit form that is easy to administer and provides uniform dosage. As used herein, "dosing unit form" refers to a physically discrete unit of agent suitable for the patient to be treated. However, it should be understood that the total daily dose of compounds and compositions is determined by the attending physician within reasonable medical judgment. The specific effective dose level for any particular patient or organism depends on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, 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. As used herein, the terms "patient" or "subject" refer to an animal, preferably a mammal, and most preferably a human.

[0233] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracerebrospinally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, or drops), orally, as an oral or nasal spray, depending on the severity of the disease or condition being treated. In some embodiments, the compounds of the present invention can be administered orally or parenterally at dose levels of about 0.01 mg / kg to about 50 mg / kg daily, and preferably about 1 mg / kg to about 25 mg / kg of the subject's body weight, once or more daily to obtain the desired therapeutic effect.

[0234] 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 also contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0235] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions contained in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectable formulations.

[0236] Injectable formulations can be sterilized, for example, by filtration via a bacterial trapping filter or by incorporation of a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0237] To prolong the effects of the compounds described herein, it is generally necessary to slow their absorption from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous substances. The absorption rate of the compound depends on its dissolution rate, which in turn depends on crystal size and crystal form. Alternatively, delayed absorption of parenteral-administered compounds can be achieved by dissolving or suspending the compound in an oily medium. Injectable reservoir forms are prepared by forming microcapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolic acid. The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoester) and poly(anhydride). Reservoir-type injectable formulations are also prepared by encapsulating the compound in tissue-compatible liposomes or microemulsions.

[0238] The composition for rectal or vaginal application is preferably a suppository, which can be prepared by mixing the compound of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which is solid at ambient temperature but liquid at body temperature, thus melting and releasing the active compound in the rectal or vaginal cavity.

[0239] 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 the following substances: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants such as glycerin; d) disintegrants such as agar, calcium carbonate, potato starch or cassava starch, alginic acid, certain silicates, and sodium carbonate; e) solution delayers such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) absorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffers.

[0240] Similar solid compositions can also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or toffee and high molecular weight polyethylene glycol. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation field. They may optionally contain light-blocking agents and may also be compositions that optionally release the active ingredient in a delayed manner only or preferably in a portion of the intestine. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or toffee and high molecular weight polyethylene glycol.

[0241] The active compound can also be in a microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells such as enteric coatings, release-controlled coatings, and other coatings well known in the pharmaceutical formulation field. In such solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose, or starch. It is common practice that such dosage forms may also contain additional substances besides 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 form may also include buffers. They may optionally contain light-blocking agents and may also be compositions that, optionally, release only or preferentially the active ingredient at a site in the intestine. Examples of encapsulation compositions that can be used include polymers and waxes.

[0242] Dosage forms for topical or transdermal application of the compounds of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or, if necessary, buffers. Ophthalmic preparations, ear drops, and eye drops are also considered to be within the scope of this invention. Additionally, this invention covers the use of transdermal patches, which have the added advantage of controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing 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 providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0243] Co-administered with one or more other therapeutic agents

[0244] Depending on the specific condition or disease to be treated, additional therapeutic agents typically used to treat that condition may also be present in the compositions of the present invention. As used herein, additional therapeutic agents typically used to treat a specific disease or condition are referred to as “the disease or condition to be treated”.

[0245] In some embodiments, the present invention provides a method of treating a disclosed disease or condition, the method comprising administering to a patient in need an effective amount of the disclosed compound 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. In some embodiments, the method comprises co-administering two additional therapeutic agents. In some embodiments, the combination of the disclosed compound and one or more additional therapeutic agents works synergistically.

[0246] The compounds of the present invention can also be used in combination with known treatment methods, such as hormone administration or radiation. In some embodiments, the provided compounds are used as radiosensitizers, particularly for treating tumors that exhibit poor sensitivity to radiotherapy.

[0247] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds. Possible combination therapies may take the form of a fixed combination or may involve alternating or independent administration of the compounds of the present invention and one or more other therapeutic compounds, or a fixed combination and a combination of one or more other therapeutic compounds. Furthermore, particularly for oncology therapy, the compounds of the present invention may be administered in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or combinations thereof. Long-term therapy is also possible, such as adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments include therapies to maintain the patient's condition after tumor regression, or even chemopreventive therapy, for example, in at-risk patients.

[0248] One or more other therapeutic agents may be administered separately from the compounds or compositions of the present invention as part of a multi-dose regimen. Alternatively, one or more other therapeutic agents may be part of a single dosage form, mixed together with the compounds of the present invention in a single composition. If administered in a multi-dose regimen, one or more other therapeutic agents and the compounds or compositions of the present invention may be administered simultaneously, sequentially, or to each other over a period of time (e.g., to each other 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). In some embodiments, one or more other therapeutic agents and the compounds or compositions of the present invention are administered as a multi-dose regimen over intervals greater than 24 hours.

[0249] As used herein, the terms "combination," "combined," and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the invention. For example, compounds of the invention may be administered simultaneously or sequentially with one or more other therapeutic agents in separate unit formulations or together in a single unit formulation. Thus, the present invention provides a single unit formulation comprising the compound of the invention, one or more other therapeutic agents, and a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0250] The amounts of the compounds of the invention, which can be combined with carrier materials to produce a single dosage form, and one or more other therapeutic agents (in those compositions comprising additional therapeutic agents as described above) vary depending on the host being treated and the specific administration method. Preferably, the compositions of the invention should be formulated such that a dose of the compounds of the invention between 0.01 and 100 mg / kg body weight / day can be administered.

[0251] In those compositions containing one or more other therapeutic agents, the other therapeutic agents and the compound of the present invention can act synergistically. Therefore, the amount of the other therapeutic agent in such compositions can be less than that required in a single therapy using only that therapeutic agent. In such compositions, a dose of one or more other therapeutic agents between 0.01 and 1,000 mg / kg body weight / day can be administered.

[0252] The amount of one or more other therapeutic agents present in the compositions of the present invention may not exceed the amount normally administered in a composition containing the therapeutic agent as the sole active agent. Preferably, the amount of one or more other therapeutic agents in the compositions disclosed in the present invention is in the range of about 50% to 100% of the amount normally present in a composition containing the agent as the sole active agent. In some embodiments, one or more other therapeutic agents are administered at a dose of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the normal administration amount of the agent. As used herein, the phrase “normal administration” means the amount of an FDA-approved therapeutic agent approved for administration according to the FDA label instructions.

[0253] The compounds of the present invention or pharmaceutical compositions thereof may also be incorporated into compositions for coating implantable medical devices such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents have been used to overcome restenosis (re-stenosis of the vessel wall after injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the devices with a pharmaceutically acceptable composition containing a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the invention.

[0254] Other exemplary therapeutic agents

[0255] In some embodiments, one or more other therapeutic agents are poly-ADP-ribose polymerase (PARP) inhibitors. In some embodiments, the PARP inhibitor is selected from olaparib (…). AstraZeneca); Lucapani ( Clovis Oncology); Nirapani Tesaro); taporabani (MDV3800 / BMN 673 / LT00673, Medivation / Pfizer / Biomarin); velipanib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

[0256] In some embodiments, one or more other therapeutic agents are histone deacetylase (HDAC) inhibitors. In some embodiments, the HDAC inhibitor is selected from vorinostat (Vorinostat). Merck); Romidesin Celgene); Papirostabrine ( Novartis; Belistar ( Spectrum Pharmaceuticals); Entecavir (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and Chidamide ( HBI-8000 (Chipscreen Biosciences, China).

[0257] In some embodiments, one or more other therapeutic agents are CDK inhibitors, such as CDK4 / CDK6 inhibitors. In some embodiments, the CDK4 / 6 inhibitor is selected from palbociclib (…). Pfizer); Rebosini Novartis; Abesil (Ly2835219, Eli Lilly); and Trilasili (G1T28, G1Therapeutics).

[0258] In some embodiments, one or more other therapeutic agents are phosphatidylinositol 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from edrexib (…). Gilead, avocadolixib (BYL719, Novartis), tasselixib (GDC-0032, Genentech / Roche); pitilixib (GDC-0941, Genentech / Roche); copanlixib (BAY806946, Bayer); duvelixib (formerly known as IPI-145, Infinity Pharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202 (formerly known as RP5230, TG Therapeutics).

[0259] In some embodiments, one or more other therapeutic agents are platinum-based therapeutic agents, also known as platinum compounds. Platinum compounds cause DNA cross-linking, thereby inhibiting DNA repair and / or DNA synthesis, primarily in rapidly proliferating cells such as cancer cells. In some embodiments, the platinum-based therapeutic agent is selected from cisplatin (…). Bristol-Myers Squibb); Carboplatin Bristol-Myers Squibb; and Teva; pfizer); oxaliplatin ( Sanofi-Aventis); Nidaplatin ( Shionogi, Pyrplatin (Poniard Pharmaceuticals); and Saplatin (JM-216, Agenix).

[0260] In some embodiments, one or more other therapeutic agents are taxane compounds that cause disruption of microtubules essential for cell division. In some embodiments, the taxane compound is selected from paclitaxel (Paclitaxel). Bristol-Myers Squibb), Dorcetsay ( Sanofi-Aventis; Sun Pharmaceutical), albumin-bound paclitaxel ( Abraxis / Celgene), Cabazitaxel ( Sanofi-Aventis) and SID530 (SK Chemicals, Co.) (NCT00931008).

[0261] In some implementations, 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.

[0262] In some implementations, the nucleoside inhibitor is selected from trabectedin (a guanidine alkylating agent). Janssen Oncology), nitrogen mustard (alkylating agent), Aktelion Pharmaceuticals); Vincristine ( Eli Lilly; Teva Pharmaceuticals; Talon Therapeutics); Temozolomide (an alkylating agent, a prodrug of 5-(3-methyltriazine-1-yl)-imidazol-4-carboxamide (MTIC)). Merck); Cytarabine injection (ara-C, an antimetabolite cytidine analog, Pfizer); Lomustine (alkylating agent, Bristol-Myers Squibb; NextSource Biotechnology); Azacitidine (a pyrimidine nucleoside analog of cytidine, Celgene); Homoharringtonine (omacetaxine mepesuccinate) (homocynotrine) (protein synthesis inhibitor) Teva Pharmaceuticals); Erwinia chrysanthemi asparaginase (an enzyme used to consume asparagine). Lundbeck; EUSA Pharma); Eribulin Mesylate (microtubule inhibitor, tubulin-based antimitotic agent, Eisai); Cabazitaxel (microtubule inhibitor, tubulin-based antimitotic agent). Sanofi-Aventis; capecitabine (thymidine synthase inhibitor) Genentech); Bendamustine (a bifunctional nitrogen mustard derivative, believed to form interstrand DNA crosslinks) Cephalon / Teva); Ixaspiron (a semi-synthetic analog of epothilone B, a microtubule inhibitor, a tubulin-based antimitotic agent). Bristol-Myers Squibb); Nerapine (a prodrug of deoxyguanosine analogue, a nucleoside metabolism inhibitor) Novartis); Clofarabine (a prodrug of ribonucleotide reductase inhibitors and a competitive inhibitor of deoxycytidine). Sanofi-Aventis; and trifluuridine and tipiracil (thymidine nucleoside analogs and thymidine phosphorylase inhibitors). Taiho Oncology).

[0263] In some embodiments, one or more other therapeutic agents are kinase inhibitors or VEGF-R antagonists. Approved VEGF inhibitors and kinase inhibitors that can be used in this invention include: the anti-VEGF monoclonal antibody bevacizumab (… Genentech / Roche); anti-VEGFR-2 antibody ramucirumab ( Eli Lilly) and aflibercept (also known as VEGF trap) Regeneron / Sanofi). VEGFR inhibitors, such as regorafenib ( Bayer); Van der Tanes ( AstraZeneca; Axitinib Pfizer); and lenvatinib ( Eisai); Raf inhibitors, such as sorafenib ( Bayer AG and Onyx); Darafini ( Novartis; and Vermofini ( Genentech / Roche); MEK inhibitors, such as cobimetinib ( Exelexis / Genentech / Roche); Trametinib ( Novartis; Bcr-Ab1 tyrosine kinase inhibitors, such as imatinib ( Novartis; Nilotinib Novartis; Dasatinib Bristol Myers Squibb); Bosutinib Pfizer); and ponatinib (Pfizer); Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such as gefitinib ( AstraZeneca; Erlotinib Genentech / Roche / Astellas); Lapatinib ( Novartis; Afatinib Boehringer Ingelheim); osimertinib (targeting activated EGFR, AstraZeneca; and brigatinib ( Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozantinib ( Exelexis; and multi-kinase inhibitors, such as sunitinib (Exelexis); ... Pfizer); Pazopanib Novartis; ALK inhibitors, such as crizotinib ( Pfizer); Ceritinib Novartis; and alectinib ( Genentech / Roche); Bruton's tyrosine kinase inhibitors, such as ibrutinib ( Pharmacyclics / Janssen); and Flt3 receptor inhibitors, such as midostaurin ( Novartis).

[0264] Other kinase inhibitors and VEGF-R antagonists under development and applicable to this invention include tivozanib (Aveo Pharmaecuticals); vatalanib (Bayer / Novartis); lucitinib (Clovis Oncology); duvitinib (TKI258, Novartis); chiauanib (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories); neratinib (HKI-272, Puma Biotechnology); and ladotinib. IY5511, I1-Yang Pharmaceuticals, South Korea; Ruxotetinib ( Incyte Corporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo); and motishanib (Amgen / Takeda).

[0265] In some embodiments, one or more other therapeutic agents are mTOR inhibitors that inhibit cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitor is everolimus (…). Novartis; Tauromus ( Pfizer); and Sirolimus ( Pfizer).

[0266] In some embodiments, one or more other therapeutic agents are proteasome inhibitors. Approved proteasome inhibitors that can be used in this invention include bortezomib (…). Takeda); Carfilzomi ( Amgen); and Ishazomi (ixazomib) Takeda.

[0267] In some embodiments, 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 their receptor (EGFR). Approved PDGF antagonists that can be used in this invention include olaratumab (…). Eli Lilly). Approved EGFR antagonists that can be used in this invention include cetuximab ( Eli Lilly); Nescilimumab ( Eli Lilly), Parlimumab ( Amgen); and osimertinib (targeting activated EGFR, AstraZeneca).

[0268] In some embodiments, one or more other therapeutic agents are aromatase inhibitors. In some embodiments, the aromatase inhibitor is selected from exemestane (…). Pfizer); Anazol ( AstraZeneca and letrozole (Fluoron) Novartis).

[0269] In some embodiments, one or more other therapeutic agents are antagonists of the hedgehog pathway. Approved hedgehog pathway inhibitors that can be used in this invention include sodegib (…). Sun Pharmaceuticals); and Vimodil ( Genentech, both are used to treat basal cell carcinoma.

[0270] In some embodiments, one or more other therapeutic agents are folic acid inhibitors. Approved folic acid inhibitors that can be used in this invention include pemetrexed (…). Eli Lilly).

[0271] In some embodiments, one or more other therapeutic agents are CC chemokine receptor 4 (CCR4) inhibitors. Investigative CCR4 inhibitors that may be used in this invention include mogamulizumab (…). Kyowa Hakko Kirin, Japan).

[0272] In some embodiments, one or more other therapeutic agents are isocitrate dehydrogenase (IDH) inhibitors. IDH inhibitors under investigation that may be used in this invention include AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); and IDH305 (Novartis, NCT02987010).

[0273] In some embodiments, one or more other therapeutic agents are arginase inhibitors. Investigative arginase inhibitors that may be used in this invention include AEB 1102 (pegylated recombinant arginase, AegleaBiotherapeutics), which is being investigated in a Phase 1 clinical trial for acute myeloid leukemia and myelodysplastic syndromes (NCT02732184) and solid tumors (NCT02561234); and CB-1158 (Calithera Biosciences).

[0274] In some embodiments, one or more other therapeutic agents are glutaminase inhibitors. Investigative glutaminase inhibitors that may be used in this invention include CB-839 (Calithera Biosciences).

[0275] In some embodiments, 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 and can be used in this invention include rituximab (…). Genentech / BiogenIdec); Ophamumab (anti-CD20, GlaxoSmithKline); Obituzumab (anti-CD20, Genentech), imbumomab (anti-CD20 and yttrium-90), Spectrum Pharmaceuticals); Daremumab (anti-CD38, Janssen Biotech), Dinutuximab (anti-glycolipid GD2) United Therapeutics); Trastuzumab (anti-HER2, Genentech); Ado-trastuzumab emtansine (anti-HER2, fused to emtansine), Genentech); and pertuzumab (anti-HER2, Genentech); and brentuximab vedotin (an anti-CD30 drug conjugate). Seattle Genetics.

[0276] In some embodiments, one or more other therapeutic agents are topoisomerase inhibitors. Approved topoisomerase inhibitors that can be used in this invention include irinotecan (…). Merrimack Pharmaceuticals); Topotecan GlaxoSmithKline). Topoisomerase inhibitors under investigation that can be used in this invention include pisacocelen (…). CTI Biopharma).

[0277] In some embodiments, 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 this invention include venetoclax (…). AbbVie / Genentech); and blinatumomab (… Amgen). Other therapeutic agents targeting apoptosis proteins that have undergone clinical trials and can be used in this invention include navitoclax (ABT-263, Abbott) and BCL-2 inhibitors (NCT02079740).

[0278] In some embodiments, one or more other therapeutic agents are androgen receptor inhibitors. Approved androgen receptor inhibitors that can be used in this invention include enzalutamide (…). Astellas / Medivation); approved androgen synthesis inhibitors include abiraterone (… Centocor / Ortho); approved gonadotropin-releasing hormone (GnRH) receptor antagonist (degaralix, Ferring Pharmaceuticals).

[0279] In some embodiments, one or more other therapeutic agents are selective estrogen receptor modulators (SERMs) that interfere with estrogen synthesis or activity. Approved SERMs that can be used in this invention include raloxifene (…). EliLilly).

[0280] In some implementations, one or more other therapeutic agents are bone resorption inhibitors. An approved therapeutic agent for inhibiting bone resorption is denosumab (…). Amgen is an antibody that binds to RANKL and prevents binding to its receptor RANK, found on the surface of osteoclasts, their precursors, and osteoclast-like giant cells, which mediates bone pathology in solid tumors with bone metastases. Other approved therapeutic agents that inhibit bone resorption include bisphosphonates, such as zoledronic acid (…). Novartis).

[0281] In some embodiments, one or more other therapeutic agents are inhibitors of the interaction between two primary p53 repressor proteins, MDMX and MDM2. Investigative p53 repressor inhibitors that can be used in this invention include ALRN-6924 (Aileron), a binding peptide that equivalently binds to and disrupts the interaction between MDMX and MDM2 with p53. ALRN-6924 is currently being evaluated in clinical trials for the treatment of AML, advanced myelodysplastic syndromes (MDS), and peripheral T-cell lymphoma (PTCL) (NCT02909972; NCT02264613).

[0282] In some embodiments, one or more other therapeutic agents are inhibitors of transforming growth factor β (TGF-beta or TGFβ). Inhibitors of the TGF-β protein under investigation that may be used in this invention include NIS793 (Novartis), an anti-TGF-β antibody clinically tested for the treatment of various cancers, including breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer (NCT 02947165). In some embodiments, the inhibitor of the TGF-β protein is fusomumab (GC1008; Sanofi-Genzyme), which is being investigated for use in melanoma (NCT00923169); renal cell carcinoma (NCT00356460); and non-small cell lung cancer (NCT02581787). Additionally, in some embodiments, another therapeutic agent is a TGF-β trap, as described by 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 known as 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-β receptor II, which acts as a TGFβ “trap.”

[0283] In some implementations, one or more other therapeutic agents are selected from glembatumumab vedotin-methylaurestatin E (MMAE) (Celldex), which is an anti-glycoprotein NMB (gpNMB) antibody (CR011) linked to the cytotoxic MMAE. gpNMB is a protein overexpressed by various tumor types associated with the metastatic ability of cancer cells.

[0284] In some embodiments, one or more other therapeutic agents are antiproliferative compounds. Such antiproliferative compounds include, but are not limited to, aromatase inhibitors; anti-estrogens; 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 the activity of protein or lipid kinases and other anti-angiogenic compounds; compounds that target, reduce, or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparinoid inhibitors; Ras oncogenic isotype inhibitors; telomerase inhibitors; proteasome inhibitors; compounds used to treat hematologic malignancies; compounds that target, reduce, or inhibit Flt-3 activity; Hsp90 inhibitors, such as those from Conforma. Therapeutics' 17-AAG (17-allylaminogeldemycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldemycin, NSC707545), IPI-504, TEMODAL CNF1010, CNF2024, CNF1010; temozolomide Kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamiprid / chlorpromazine from CombinatoRx; MEK inhibitors, such as ARRY142886 from Array BioPharma, AZd6244 from AstraZeneca, PD181461 from Pfizer, and hydroxylamine.

[0285] As used herein, the term "aromatase inhibitor" refers to compounds that inhibit estrogen production (e.g., the conversion of substrates androstenedione and testosterone into estrone and estradiol, respectively). This term includes, but is not limited to, steroids (especially atemetane, exemestane, and formestane), and particularly nonsteroidal agents (especially aminoglutethimide, roglethimide, pyrglutethimide, tralostertan, testrolide, ketoconazole, vortexilazole, faucizoxin, anastrozole, and letrozole). Exemestane is marketed under the trade name AROMASIN. TM Sales. Formistan is marketed under the trade name Lentaron. TM Sales. Fazodazole is marketed under the brand name AFEMA. TM Anastrozole is sold under the brand name ARIMIDEX. TM Sales. Letrozole is marketed under the brand name FEMARA. TMor FEMAr TM Sales. Ammonia luminetide is marketed under the trade name ORIMETEN. TM Sales. The combinations of the present invention, comprising chemotherapeutic agents that are aromatase inhibitors, are particularly useful for treating hormone receptor-positive tumors, such as breast tumors.

[0286] As used herein, the term "anti-estrogenic drug" refers to compounds that antagonize 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 marketed under the brand name NOLVADEX. TM Sales. Raloxifene hydrochloride is marketed under the brand name EVISTA. TM Sales. Fluvestracum can be sold under the brand name FASLODEX. TM Application. The combinations of the present invention, comprising chemotherapeutic agents that are anti-estrogens, are particularly useful for treating estrogen receptor-positive tumors, such as breast tumors.

[0287] As used herein, the term "anti-androgen" refers to any substance capable of inhibiting the biological effects of androgens, and includes, but is not limited to, bicalutamide (CASODEX). TM As used herein, the term "goserelin agonist" includes, but is not limited to, abaric, goserelin, and goserelin acetate. Goserelin may be marketed under the brand name ZOLADEX. TM Apply.

[0288] As used herein, the term "topoisomerase I inhibitor" includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-nitrocamptothecin, and the macromolecular camptothecin conjugate PNU-166148. Irinotecan may be marketed, for example, under the trade name CAMPTOSAR. TM Topotecan is administered through sales channels. It is marketed under the brand name HYCAMPTIN. TM Sale.

[0289] As used herein, the term "topoisomerase II inhibitor" includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations such as CAELYX). TM ), daunorubicin, epirubicin, idarubicin, and nemorubicin; anthraquinones mitoxantrone and loxoantrone; and podophyllotoxins etoposide and teniposide. Etoposide is marketed under the brand name ETOPOPHOS. TM Sales. Teniposide is marketed under the brand name VM 26-Bristol. Doxorubicin is marketed under the brand name ACRIBLASTIN. TM Or ADRIAMYCIN TM Sales. The product name is Farmorubicin. TM Sales. Itabizin is sold under the trade name ZAVEDOS.TM Mitoxantrone is sold under the trade name NOVANTRON. TM Sale.

[0290] The term "microtubule activator" encompasses microtubule stabilizers, microtubule destabilizers, and microtubule polymerization inhibitors, including but not limited to taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vinblastine sulfate, and vinorelbine; discermolide; colchicine and epothilone and their derivatives. Paclitaxel is marketed under the trade name TAXOL. TM Sales. Dorcetazetta is marketed under the brand name TAXOTERE. TM Sales. Vinblastine sulfate is sold under the trade name VINBLASTIN Rp. TM Sales. Vincristine sulfate is marketed under the trade name FARMISTIN. TM Sale.

[0291] As used herein, the term "alkylating agent" includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is marketed under the trade name CYCLOSTIN. TM Ifosfamide is sold under the trade name HOLOXAN. TM Sale.

[0292] The term "histone deacetylase inhibitor" or "HDAC inhibitor" refers to compounds that inhibit histone deacetylases and have antiproliferative activity. This includes, but is not limited to, succinyl aniline isohydroxamic acid (SAHA).

[0293] The term "antitumor antimetabolites" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edarax, and folic acid antagonists such as pemetrexed. Capecitabine is marketed under the brand name XELODA. TM Sales. Gemcitabine is sold under the brand name GEMZAR. TM Sale.

[0294] As used herein, the term "platinum compound" includes, but is not limited to, carboplatin, cisplatin, and oxaliplatin. Carboplatin may be marketed, for example, under the trade name CARBOPLAT. TM Oxaliplatin can be applied through sales channels, for example, under the brand name ELOXATIN. TM The form of sales is applied.

[0295] As used herein, the terms “compounds that target / reduce the activity of protein or lipid kinases”; or protein or lipid phosphatase activity; or “other anti-angiogenic compounds” include, but are not limited to, protein tyrosine kinase and / or serine and / or threonine kinase inhibitors or lipid kinase inhibitors, such as a) compounds that target, reduce or inhibit platelet-derived growth factor receptor (PDGFR) activity, such as compounds that target, reduce or inhibit PDGFR activity, especially compounds that inhibit PDGF receptors, such as N-phenyl-2-pyrimidin-amine derivatives, such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target, reduce or inhibit fibroblast growth factor receptor (FGFR) activity; c) compounds that target, reduce or inhibit insulin-like growth factor receptor I (IGF-IR) activity, such as compounds that target, reduce or inhibit IGF-IR activity, especially compounds that inhibit the kinase activity of IGF-I receptors or antibodies that target the extracellular domains of IGF-I receptors or their growth factors; d) compounds that target, reduce or inhibit Compounds targeting, reducing, or inhibiting the activity of the Trk receptor tyrosine kinase family, or hepatin B4 inhibitors; e) compounds targeting, reducing, or inhibiting the activity of the AxI receptor tyrosine kinase family; f) compounds targeting, reducing, or inhibiting the activity of the Ret receptor tyrosine kinase family; g) compounds targeting, reducing, or inhibiting the activity of the Kit / SCFR receptor tyrosine kinase family, such as imatinib; h) compounds targeting, reducing, or inhibiting the activity of the C-kit receptor tyrosine kinase family, which is part of the PDGFR family, such as compounds targeting, reducing, or inhibiting the activity of the c-Kit receptor tyrosine kinase family, especially compounds inhibiting the c-Kit receptor, such as imatinib; i) compounds targeting, reducing, or inhibiting the activity of c-Ab1 family members, their gene fusion products (e.g., BCR-Ab1 kinase), and mutants, such as compounds targeting, reducing, or inhibiting the activity of c-Ab1 family members and their gene fusion products, such as N-phenyl-2-pyrimidin-amine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from Parke Davis; or dasatinib (BMS-354825); j) compounds that target, reduce, or inhibit the activity of Raf family members of protein kinase C (PKC) and serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK, and TEC family members and / or cyclin-dependent kinase family (CDK) members, including astrone derivatives such as midostaurin; other examples of compounds include UCN-01, safungo, BAY 43-9006, bryostatin 1, perifoxine; imofoxine;RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531 / LY379196; isochinoline compounds; FTI; PD184352 or QAN697 (P13K inhibitor) or AT7519 (CDK inhibitor); k) compounds that target, reduce or inhibit the activity of protein tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC); TM ) or tyrosine phosphorylation inhibitors (tyrphostin), such as tyrosine phosphorylation inhibitor A23 / RG-50810; AG 99; tyrosine phosphorylation inhibitor AG213; tyrosine phosphorylation inhibitor AG 1748; tyrosine phosphorylation inhibitor AG 490; tyrosine phosphorylation inhibitor B44; tyrosine phosphorylation inhibitor B44(+) enantiomer; tyrosine phosphorylation inhibitor AG 555; AG 494; tyrosine phosphorylation inhibitor AG 556, AG957 and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-adaphostin; NSC 680410, adaphostin); 1) Targeting, reducing or inhibiting the epidermal growth factor receptor tyrosine kinase family (EGFR1 Compounds containing ErbB2, ErbB3, and ErbB4 as homodimers or heterodimers and their mutants (such as compounds that target, reduce, or inhibit the activity of the epidermal growth factor receptor family), especially compounds, proteins, or antibodies that inhibit members of the EGF receptor tyrosine kinase family (such as EGF receptor, ErbB2, ErbB3, and ErbB4) or bind to EGF or EGF-related ligands; CP 358774, ZD 1839, ZM 105180; trastuzumab (HERCEPTIN) TM Cetuximab (ERBITUX) TM(i) Iressa, Tarceva, OSI-774, C1-1033, 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, reduce or inhibit c-Met receptor activity, such as compounds that target, reduce or inhibit c-Met activity, especially compounds that inhibit c-Met receptor kinase activity, or antibodies that target the extracellular domain of c-Met or bind HGF; n) Compounds that target, reduce or inhibit the activity of one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan-JAK) kinases, 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, reduce, or inhibit the kinase activity of PI3 kinase (PI3K), including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and edroxysibulin; and q) compounds that target, reduce, or inhibit the activity of hedgehog protein (Hh) or smoothed receptors. Compounds that act as signal transduction agents in the receptor (SMO) pathway, including but not limited to cycloparamine, vemodigine, itraconazole, erismodegib, and IPI-926 (sarediazole).

[0296] As used herein, the term "PI3K inhibitor" includes, but is not limited to, compounds that have inhibitory activity against one or more enzymes in the phosphatidylinositol-3-kinase family, including but not limited to PI3Kα, PI3Kγ, PI3Kμ, 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 that can be used in this invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, bupanicillin, pictrelisib, PF-4691502, BYL-719, datolis, XL-147, XL-765, and edrithib.

[0297] As used herein, the term "Bcl-2 inhibitor" includes, but is not limited to, compounds with inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737, apogospermum, Ascenta'span-Bcl-2 inhibitors, curcumin (and its analogues), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Gennathys (G3139), HA14-1 (and its analogues; see WO2008118802), navittox (and its analogues; see US7390799), NH-1 (Shenayng Pharmaceutical University), ocabacla (and its analogues; see WO2004106328), S-001 (Gloria Pharmaceuticals), TW series compounds (University of Michigan), and venetox. In some embodiments, Bcl-2 inhibitors are small molecule therapeutic agents. In some implementations, Bcl-2 inhibitors are peptidomimetic.

[0298] As used herein, the term "BTK inhibitor" 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.

[0299] As used herein, the term "SYK inhibitor" 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, Excellair, PRT-062607, and futtatinib.

[0300] Further examples of BTK inhibitory compounds and conditions that can be treated by combining such compounds with the compounds of the present invention can be found in WO2008039218 and WO2011090760, the entire contents of which are incorporated herein by reference.

[0301] Further examples of SYK inhibitory compounds and conditions that can be treated by combining such compounds with the compounds of the present invention can be found in WO2003063794, WO2005007623 and WO2006078846, the entire contents of which are incorporated herein by reference.

[0302] Further examples of PI3K inhibitory compounds and conditions that can be treated by combining such compounds 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.

[0303] Further examples of JAK inhibitory compounds and conditions that can be treated by combining such compounds with the compounds of the present invention can be found in WO2009114512, WO2008109943, WO2007053452, WO2000142246 and WO2007070514, the entire contents of which are incorporated herein by reference.

[0304] Other anti-angiogenic compounds include those with an alternative mechanism of activity, for example, independent of protein or lipid kinase inhibition, such as thalidomide. TM ) and TNP-470.

[0305] Examples of proteasome inhibitors that can be used in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0306] Compounds that target, reduce, or inhibit the activity of protein or lipid phosphatases are, for example, inhibitors of phosphatase 1, phosphatase 2A, or CDC25, such as okadaic acid or its derivatives.

[0307] Compounds that induce cell differentiation include, but are not limited to, retinoic acid, α-, γ- or δ-tocopherol, or α-, γ- or δ-tocotrienol.

[0308] As used herein, cyclooxygenase inhibitors include, but are not limited to, Cox-2 inhibitors, 5-alkyl-substituted 2-arylaminophenylacetic acid and their derivatives, such as celecoxib. TM ), Vioxx TM ), atecoxib, vardicoxib or 5-alkyl-2-arylaminophenylacetic acid, such as 5-methyl-2-(2′-chloro-6′-fluoroaniline)phenylacetic acid, luminicoxib.

[0309] As used herein, the term "bisphosphonate" includes, but is not limited to, etidronic acid, clodronic acid, teludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid. Etridonic acid is marketed under the trade name DIDRONEL. TM Sales. Chlorphosphine is marketed under the trade name BONEFOS. TM Sales. Tiludronic acid is marketed under the brand name Skelid. TM Sales. Pamidronate is marketed under the brand name AREDIA. TM Sales. Alendronate is sold under the brand name FOSAMAX. TM Ibandronic acid is sold under the trade name BONDRANAT. TM Risedronate is sold under the trade name ACTONEL. TM Sales. Zoledronic acid is marketed under the trade name ZOMETA. TM Sales. The term "mTOR inhibitor" refers to compounds that inhibit the mammalian target of rapamycin (mTOR) and have antiproliferative activity, such as sirolimus. Everolimus (CERTICAN TM ), CCI-779 and ABT578.

[0310] As used herein, the term "heparinase inhibitor" refers to a compound that targets, reduces, or inhibits the degradation of heparin sulfate. This term includes, but is not limited to, PI-88. As used herein, the term "biological response modifier" refers to lymphokines or interferons.

[0311] As used in this article, "inhibitors of Ras oncogenic isotypes," such as H-Ras, K-Ras, or N-Ras, refers to compounds that target, reduce, or inhibit the oncogenic activity of Ras; for example, "farnesyltransferase inhibitors" such as L-744832, DK8G557, or R115777 (ZARNESTRA) TM 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 those that inhibit telomerase receptors, such as telomestatin.

[0312] As used herein, the term "methionine aminopeptidase inhibitor" refers to a compound that targets, reduces, or inhibits the activity of methionine aminopeptidase. Compounds that target, reduce, or inhibit methionine aminopeptidase activity include, but are not limited to, bengamide or its derivatives.

[0313] As used herein, the term "proteasome inhibitor" refers to compounds that target, reduce, or inhibit proteasome activity. Compounds that target, reduce, or inhibit proteasome activity include, but are not limited to, bortezomib (VELCADE). TM ) and MLN341.

[0314] As used herein, the term “matrix metalloproteinase inhibitor” or “MMP” inhibitor includes, but is not limited to, collagen peptide-like and non-peptide-like inhibitors, tetracycline derivatives, such as the oxime peptide-like inhibitor palmastat and its oral bioavailable analogues malimasat (BB-2516), pruristat (AG3340), metastat (NSC683551) BMS-279251, BAY 12-9566, TAA211, MMI270B, or AAJ996.

[0315] As used herein, the term "compounds for the treatment of hematologic malignancies" includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, reduce or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R); interferons, 1-β-D-ara-cytosine 1-ara-c and bisulfan; and ALK inhibitors, which are compounds that target, reduce or inhibit anaplastic lymphoma kinase.

[0316] Compounds that target, reduce, or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R), especially compounds, proteins, or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, astrocytocin derivatives, SU11248, and MLN518.

[0317] As used herein, the term "HSP90 inhibitor" includes, but is not limited to, compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90; and HSP90 client proteins that degrade, target, reduce, or inhibit via the ubiquitin-proteasome pathway. Compounds that target, reduce, or inhibit the intrinsic ATPase activity of HSP90, particularly compounds, proteins, or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldmycin (17AAG), geldmycin derivatives; other geldmycin-related compounds; rhizocarpine and HDAC inhibitors.

[0318] As used in this article, the term "antiproliferative antibody" includes, but is not limited to, trastuzumab (HERCEPTIN). TM Trastuzumab-DM1, Erbitux, Bevacizumab (AVASTIN) TM ), rituximab PRO64553 (anti-CD40) and 2C4 antibodies. Antibodies refer to complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two complete antibodies, and antibody fragments, as long as they exhibit the desired biological activity.

[0319] For the treatment of acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia therapies, particularly with therapies used to treat AML. Specifically, the compounds of the present invention can be administered in combination with, for example, farnesyltransferase inhibitors and / or other drugs that can be used to treat AML, such as daunorubicin, doxorubicin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatin, and PKC412.

[0320] Other anti-leukemia compounds include, for example, Ara-C (a pyrimidine analog), a ′-α-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine analogs of hypoxanthine, 6-mercaptopurine (6-MP), and fludarabine phosphate. Compounds that target, reduce, or inhibit the activity of histone deacetylase (HDAC) inhibitors, such as sodium butyrate and succinyl aniline isohydroxamic acid (SAHA), inhibit the activity of enzymes called histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly known as FR901228), trichostatin A, and compounds disclosed in US 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-acrylamide or pharmaceutically acceptable salts thereof and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-acrylamide or pharmaceutically acceptable salts thereof, particularly lactates. Somatostatin receptor antagonists, as used herein, refer to compounds that target, treat, or inhibit somatostatin receptors, such as octreotide and SOM230. Tumor cell damage methods refer to methods such as ionizing radiation. The term “ionizing radiation” as used above and below means ionizing radiation in the form of electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiotherapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, edited by Devita et al., 4th ed., Vol. 1, pp. 248–275 (1993).

[0321] This also includes EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressants that regulate lymphocyte recirculation, such as FTY720. The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs, including but not limited to fludarabine and / or cytarabine (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C to combat ALL), and / or pentostatin. Ribonucleotide reductase inhibitors are particularly hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0322] This also specifically includes compounds, proteins, or monoclonal antibodies against VEGF, such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or its pharmaceutically acceptable salts, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; ANGIOSTATIN TM ;ENDOSTATIN TM ; anthranilamide; ZD4190; Zd6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as rhuMAb and RHUFab, VEGF aptamers such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgG1 antibodies, angiozyme (RPI 4610) and bevacizumab (AVASTIN) TM ).

[0323] As used in this article, photodynamic therapy refers to the treatment or prevention of cancer using certain chemicals called photosensitizing compounds. Examples of photodynamic therapy include the use of compounds such as VISUDYNE. TM Treatment with sodium porphyrin.

[0324] As used in this article, vasopressor steroids refer to compounds that block or inhibit angiogenesis, such as anechodine, triamcinolone, hydrocortisone, 11-α-epiocotisol, todoxacin, 17α-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone, and dexamethasone.

[0325] Implants containing corticosteroids refer to compounds such as fluocinolone acetonide and dexamethasone.

[0326] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormone compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or hybrid compounds or compounds with other or unknown mechanisms of action.

[0327] The structure of an active compound identified by its code number, generic name, or trade name can be taken from the actual version of the standard outline "The Merck Index" or from databases such as international patents (e.g., IMS World Publications).

[0328] Exemplary immunotumor agents

[0329] In some embodiments, one or more other therapeutic agents are immunotumor agents. As used herein, the term "immunotumor agent" refers to an agent that effectively enhances, stimulates, and / or upregulates the immune response in a subject. In some embodiments, the immunotumor agent has a synergistic effect with the administration of the compounds of the present invention in the treatment of cancer.

[0330] Immunotumor agents can be, for example, small molecule drugs, antibodies, or biological or small molecule agents. Examples of biological immunotumor 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-derived.

[0331] In some implementations, the immunotumor agent is either an agonist that stimulates (including co-stimulates) receptors or an antagonist that inhibits (including co-inhibitory) signals on T cells, both of which result in amplification of antigen-specific T cell responses.

[0332] Some stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory 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 co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to members of the homologous TNF receptor family, 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, and RANK. L, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR 3. EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0333] In some implementations, the immunotumor 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.

[0334] In some embodiments, the combination of the compounds of the present invention and the immunotumor agents can stimulate a T cell response. In some embodiments, the immunotumor agents are: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galactagogue 9, CEACAM-1, BTLA, CD69, galactagogue 1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; or (ii) agonists of proteins that stimulate 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.

[0335] In some embodiments, the immunotumor agent is an antagonist of inhibitory receptors on NK cells or an agonist of activating receptors on NK cells. In some embodiments, the immunotumor agent is an antagonist of KIRs, such as lirilumab.

[0336] In some implementations, the immunotumor 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).

[0337] In some embodiments, the immunotumor agent is selected from agonists that connect to positive co-stimulatory receptors; blockers that attenuate signal transduction via inhibitory receptors; antagonists; and one or more agents that systemically increase the frequency of antitumor T cells; agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor binding (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., dacrolimus) or depleting them via in vitro anti-CD25 beads), inhibiting metabolic enzymes such as IDO, or reversing / preventing T cell energy or depletion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0338] 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 trimemumab.

[0339] In some embodiments, the immunotumor agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immunotumor agent is an antibody or its antigen-binding moiety 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; WO2012 / 145493). In some embodiments, the immunotumor agent may be pildizumab (CT-011). In some embodiments, the immunotumor agent is a recombinant protein, referred to as AMP-224, consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1.

[0340] 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 / 79174), or cimiprimab (REGN2810).

[0341] 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 LAG3 antibody is BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO009 / 44273).

[0342] 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 urerutumab or PF-05082566 (WO12 / 32433).

[0343] In some embodiments, the immunotumor 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).

[0344] In some implementations, the immunotumor agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some implementations, the IDO antagonist is selected from icardostat (INCB024360, Incyte); indomod (NLG-8189, NewLinkGenetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS: F001287 (Bristol-Myers Squibb); Phy906 / KD 108 (Phytoceutica); kynuronine-degrading enzyme (Kynase, Ikena Oncology, formerly known as Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0345] In some embodiments, the immunotumor agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0346] In some embodiments, the immunotumor agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO06 / 029879).

[0347] In some embodiments, the immunotumor agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonist CD40 antibody. In some embodiments, the immunotumor agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonist CD40 antibody. In some embodiments, the CD40 antibody is rukalimumab or dasizumab.

[0348] 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 varigramab.

[0349] In some implementations, the immunotumor agent is MGA271 (targeting B7H3) (WO11 / 109400).

[0350] In some implementation schemes, the immuno-oncology agents are abavoxib, adenomyumab, atorciizumab, alemtuzumab, and anatumomab. Mafenatox, aporizumab, atezolizumab, averuzumab, bonatetuzumab, BMS-936559, caputuzumab, durvalumab, icardostab, iprazumab, indomod, intozumab, ozomicin, intelumumab, ipilimumab, isarucizumab, lambrolizumab, MED14736, MPDL3280A, nivolumab, oxabutuzumab, oxcaputuzumab, olatatumab, pembrolizumab, pildizumab, rituximab, teximumab, samalizumab, or trimelimumab.

[0351] In some implementations, the immuno-oncology agent is an immunostimulant. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can deplete activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in increased numbers of tumor histology, including some tumor types not conventionally considered sensitive to immunotherapy. See, for example, Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al. (2016) Sci. Transl. Med. 8. Anti-PD-1 antibody nivolumab ( Bristol-Myers Squibb (also known as ONO-4538, MDX1 106, and BMS-936558) has shown potential to improve overall survival in patients with RCC who experience disease progression during or after prior anti-angiogenic therapy.

[0352] In some embodiments, the immunomodulatory therapeutic agent specifically induces apoptosis in tumor cells. Approved immunomodulatory therapeutic agents that can be used in this invention include pomalidomide (…). Celgene); Lenalidomide Celgene); Megaphoryl methyl butenoate ( LEO Pharma).

[0353] In some embodiments, the immunotumor agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from sipuleucel-T (…). Dendreon / Valeant Pharmaceuticals, which has been approved for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogenelaherparepvec ( BioVex / Amgen (formerly known as T-VEC) is a genetically modified oncolytic virus therapy approved for the treatment of unresectable cutaneous, subcutaneous, and nodular lesions of melanoma. In some embodiments, the immunotumor agent is selected from oncolytic virus therapies such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), an engineered thymidine kinase-(TK-)-deficient vaccinia virus expressing GM-CSF, used for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (… Oncolytics Biotech describes a respiratory enteric orphan virus (ROV) variant that does not replicate in non-RAS-activated cells in a variety of 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) (NCT01622543). 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), an adenovirus engineered to express full-length CD80 and antibody fragments specific to the T-cell receptor CD3 protein in the following diseases: ovarian cancer (NCT02028117); metastatic or advanced epithelial tumors, such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly known as Oncos), an adenovirus engineered to express GM-CSF in the following diseases: melanoma (NCT03003676); and peritoneal diseases, colorectal cancer, or ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux) GmbH, which is a vaccinia virus engineered to express β-galactosidase (β-gal) / β-glucuronidase or β-gal / human sodium iodide co-transporter (hNIS), and has been studied in peritoneal cancer metastasis (NCT01443260), fallopian tube cancer, and ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), which is an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818).

[0354] In some embodiments, the immuno-oncology agent is selected from JX-929 (SillaJen / formerly JennerexBiotherapeutics), an engineered vaccinia virus lacking TK- and vaccinia growth factor that can convert the prodrug 5-fluorocytosine into the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly known as Oncos), peptide-based immunotherapeutic agents targeting refractory RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus named Ad5 / 3-E2F-delta24-hTNFα-IRES-hIL20; and VSV-GP (ViraTherapeutics), a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express a protein designed to produce antigen-specific CD8. + Antigens that trigger T cell responses.

[0355] In some implementations, the immuno-oncology agent is a T cell engineered to express a chimeric antigen receptor, or CAR. T cells engineered to express this chimeric antigen receptor are called CAR-T cells.

[0356] A CAR has been constructed consisting of a binding domain derived from a natural ligand or a single-chain variable fragment (scFv) derived from a monoclonal antibody specific to cell surface antigens, fused to an intracellular domain that serves as a functional terminus of the T cell receptor (TCR), such as the CD3-ζ signaling domain from the TCR, which can generate an activation signal in T lymphocytes. Upon antigen binding, this type of CAR links to endogenous signaling pathways in effector cells and generates an activation signal similar to that induced by the TCR complex.

[0357] For example, in some embodiments, CAR-T cells are one of those described in U.S. Patent 8,906,682 (June et al.; incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to include an extracellular domain (such as a CD19-binding domain) having an antigen-binding domain fused to an intracellular signaling domain (such as CD3ζ) of the T cell antigen receptor complex ζ chain. When expressed in T cells, the CAR is able to redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Currently, more than 200 clinical trials are underway for the use of CAR-T in a wide range of indications. [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0358] In some implementations, the immunostimulant is an activator of retinoic acid receptor-associated orphan receptor g (RORgt). RORgt is a transcription factor that plays a key role in the differentiation and maintenance of the type 17 effector subset 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 implementations, the activator of RORgt is LYC-55716 (Lycera), which is currently being evaluated in a clinical trial for the treatment of solid tumors (NCT02929862).

[0359] In some embodiments, the immunostimulant is an agonist or activator of a toll-like receptor (TLR). Suitable TLR activators include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG that is being investigated for B-cell, follicular, and other lymphomas (NCT02254772). Agonists or activators of TLR8 that can be used in this invention include mototimod (VTX-2337, VentiRx Pharmaceuticals), which is being investigated for head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[0360] Other immuno-oncology agents that can be used in this invention include urerutumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varirucumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirerucumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monarizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; and adeliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0361] In some implementations, the immunostimulant is selected from elotuzumab, mifamurtide, agonists or activators of Toll-like receptors, and activators of RORγt.

[0362] In some embodiments, the immunostimulatory agent is recombinant human interleukin-15 (rhIL-15). rhIL-15 has been clinically tested as a therapy for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemia (NCT02689453). In some embodiments, the immunostimulator is recombinant human interleukin-12 (rhIL-12). In some embodiments, the IL-15-based immunotherapy agent is heterodimeric IL-15 (hetIL-15, Novartis / Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 (IL15: sIL-15RA) complexed with the α chain of the soluble IL-15-binding protein IL-15 receptor, which has been 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 implementations, recombinant human interleukin-12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.) (NCT02544724 or NCT02542124).

[0363] In some embodiments, the immuno-oncology agent is selected from those described in Jerry L. Adams et al., “Bigopportunities for small molecules in immuno-oncology,” Cancer Therapy 2015, Vol. 14, pp. 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 described in Table 1 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule targeting immuno-oncology targets selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule agent selected from those listed in Table 2 of Jerry L. Adams et al.

[0364] 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, pp. 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 the pathway described in Peter L. Toogood.

[0365] In some implementations, the immunotumor agent is selected from Sandra L. Ross et al., "Bispecific T cellengager". The contents of the literature described in “antibody constructs can mediate bystander tumor cell killing”, PLoS ONE 12(8): e0183390, are incorporated herein by reference in their entirety. In some embodiments, the immunotumor agent is a bispecific T-cell conjugate. Antibody constructs. In some implementations, bispecific T-cell conjugates. The antibody construct is a CD19 / CD3 bispecific antibody construct. In some implementations, a bispecific T-cell conjugate... The antibody construct is an EGFR / CD3 bispecific antibody construct. In some implementations, a bispecific T-cell conjugate... Antibody constructs activate T cells. In some implementations, bispecific T cell conjugates... The antibody construct activates T cells, which release cytokines that induce intercellular adhesion molecule-1 (ICAM-1) and FAS upregulation on bystander cells. In some embodiments, a bispecific T cell conjugate... The antibody construct activates T cells, which leads to induced bystander cell lysis. In some embodiments, the bystander cells are in a solid tumor. In some embodiments, the lysed bystander cells are in... Near activated T cells. In some embodiments, bystander cells include tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, bystander cells include EGFR-negative cancer cells. In some embodiments, the immunotumor agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immunotumor agent is ex vivo expanded tumor-infiltrating T cells. In some embodiments, the immunotumor agent is a bispecific antibody construct or chimeric antigen receptor (CAR) that directly links T cells to tumor-associated surface antigens (TAAs).

[0366] Exemplary immune checkpoint inhibitors

[0367] In some implementations, the immunotumor agent is an immune checkpoint inhibitor as described herein.

[0368] As used in this article, the term "checkpoint inhibitor" refers to agents that can be used to prevent cancer cells from evading a patient's immune system. One of the main mechanisms of antitumor immune destruction is known as "T-cell exhaustion," which is caused by prolonged exposure to antigens that lead to the upregulation of inhibitory receptors. These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune responses.

[0369] PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuators (BTLA; CD272), T-cell immunoglobulin and mucin domain 3 (Tim-3), lymphocyte activation gene 3 (Lag-3; CD223), etc., are commonly referred to as checkpoint regulators. They act as molecular "gatekeepers," allowing extracellular information to indicate whether cell cycle progression and other intracellular signaling processes should occur.

[0370] In some implementations, immune checkpoint inhibitors are antibodies against PD-1. PD-1 binds to programmed cell death 1 receptor (PD-1) to prevent the receptor from binding to the inhibitory ligand PDL-1, thereby overcoming the ability of tumors to suppress the host's antitumor immune response.

[0371] In some embodiments, the checkpoint inhibitor is a biological therapeutic agent or a 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. In some embodiments, 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 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 combinations thereof. In some embodiments, the checkpoint inhibitor is an immunostimulant, T-cell growth factor, interleukin, antibody, 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 another aspect, the vaccine is a dendritic cell (DC) vaccine.

[0372] Checkpoint inhibitors include any agent that statistically significantly blocks or inhibits inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies that bind to and block or inhibit immune checkpoint receptors or their antigen-binding fragments, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that can be targeted for blockage or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, and 2B4 (belonging to the CD2 molecule family and present in all NK, γδ, and memory CD8 molecules). +(αβ) T cell-expressed), 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, biotherapeutic agents, or small molecules that bind to and block or inhibit 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, but are not limited to, trimemumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and ipilimumab (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.

[0373] In some embodiments, the immune checkpoint inhibitor is selected from PD-1 antagonists, PD-L1 antagonists, and CTLA-4 antagonists. In some embodiments, the checkpoint inhibitor is selected from the group consisting of the following: nivolumab Ipilimumab and pembrolizumab In some embodiments, the checkpoint inhibitor is selected from nivolumab (an anti-PD-1 antibody). Bristol-Myers Squibb); Pembrolizumab (anti-PD-1 antibody) Merck); Ipilimumab (anti-CTLA-4 antibody), Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody); AstraZeneca); and atezolizumab (an anti-PD-L1 antibody). Genentech).

[0374] In some implementations, the checkpoint inhibitors are selected from the group consisting of: lanlolizumab (MK-3475), nivolumab (BMS-936558), pildilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirelurumab, IPH2101, and pembrolizumab. And trimelimumab.

[0375] In some implementations, immune checkpoint inhibitors include REGN2810 (Regeneron), an anti-PD-1 antibody tested in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pildizumab (CureTech), also known as CT-011, an antibody that binds to PD-1 in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; and avelumab (…). Pfizer / Merck KGaA, also known as MSB0010718C, is a fully human IgG1 anti-PD-L1 antibody used in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, kidney cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer; or PDR001 (Novartis), an inhibitory antibody that binds to PD-1 used in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Trimelimumab (CP-675, 206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4, which has been investigated in clinical trials for a variety of indications, including: mesothelioma, colorectal cancer, renal cell carcinoma, breast cancer, lung cancer, non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, intrahepatic metastatic cancer, 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 (NCT02694822) investigated in a phase 1 clinical trial for advanced solid tumors.

[0376] In some embodiments, the checkpoint inhibitor is an inhibitor of protein 3 (TIM-3), which contains T-cell immunoglobulin mucin. TIM-3 inhibitors that can be used in this invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody (NCT02817633) being studied in solid tumors. LY3321367 (Eli Lilly) is an anti-TIM-3 antibody (NCT03099109) being studied in solid tumors. MBG453 (Novartis) is an anti-TIM-3 antibody (NCT02608268) being studied in advanced malignant tumors.

[0377] In some embodiments, checkpoint inhibitors are inhibitors of T-cell immune receptors having Ig and ITIM domains or of TIGIT (an immune receptor on certain T cells and NK cells). TIGIT inhibitors that can be used in this invention include BMS-986207 (Bristol-Myers Squibb), an anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and an anti-TIGIT monoclonal antibody (NCT03119428).

[0378] In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene 3 (LAG-3). LAG-3 inhibitors that can be used in this invention include BMS-986016, REGN3767, and IMP321. The anti-LAG-3 antibody BMS-986016 (Bristol-Myers Squibb) is being investigated in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody and is being investigated in malignant tumors (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein that is being investigated in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[0379] Checkpoint inhibitors that can be used in this invention include OX40 agonists. OX40 agonists currently being investigated in clinical trials include PF-04518600 / PF-8600 (Pfizer), an agonistic anti-OX40 antibody for metastatic renal cell carcinoma (NCT03092856) and advanced cancer and tumors (NCT02554812); NCT05082566; GSK3174998 (Merck), an agonistic anti-OX40 antibody in a phase 1 cancer trial (NCT02528357); and MEDI0562 (Medimmune / AstraZene). Medimmune (AstraZeneca), an agonist anti-OX40 antibody, is used for advanced solid tumors (NCT02318394 and NCT02705482); MEDI6469, an agonist anti-OX40 antibody (Medimmune / AstraZeneca), is used for 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 agonist anti-OX40 antibody, is used for advanced cancer (NCT02737475).

[0380] Checkpoint inhibitors that can be used in this invention include CD137 (also known as 4-1BB) agonists. CD137 agonists being investigated in clinical trials include urtorumab (PF-05082566, Pfzer), an agonistic anti-CD137 antibody for diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and tumors (NCT02554812 and NCT05082566); urtorumab (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD137 antibody for melanoma and skin cancer (NCT02652455) and glioblastoma and glioma (NCT02658981); and CTX-471 (Compass Therapeutics), an agonistic anti-CD137 antibody for metastatic or locally advanced malignancies (NCT03881488).

[0381] Checkpoint inhibitors that can be used in this invention include CD27 agonists. CD27 agonists being investigated in clinical trials include varigramab (CDX-1127, Ce11dex Therapeutics), an agonistic anti-CD27 antibody used for squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0382] Checkpoint inhibitors that can be used in this invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists currently under investigation in clinical trials include TRX518 (Leap Therapeutics), an agonist anti-GITR antibody for malignant melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonist anti-GITR antibody for solid tumors and lymphomas (NCT 02740270); INCAGN01876 (Incyte / Agenus), an agonist anti-GITR antibody for advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonist anti-GITR antibody for solid tumors (NCT02132754); and MEDI1873 (Medimmune / AstraZeneca), an antibody containing human IgG1. An agonistic hexameric GITR ligand molecule with an Fc domain, for use in advanced solid tumors (NCT02583165).

[0383] Checkpoint inhibitors that can be used in this invention include inducible T-cell co-stimulatory factor (ICOS, also known as CD278) agonists. ICOS agonists currently being investigated in clinical trials include MEDI-570 (Medimmune), an agonistic anti-ICOS antibody for lymphoma (NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody in a phase 1 trial (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody in a phase 1 trial (NCT02904226).

[0384] Checkpoint inhibitors that can be used in this invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors currently under investigation in clinical trials include lirelurumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody used for leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma), used for myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to three domains of the long cytoplasmic tail (KIR3DL2), used for lymphoma (NCT02593045).

[0385] Checkpoint inhibitors that can be used in this invention include CD47 inhibitors that address the interaction between CD47 and signal regulatory protein α (SIRPa). CD47 / SIRPa inhibitors currently under investigation in clinical trials include ALX-148 (AlexoTherapeutics), a (SIRPa) antagonistic variant that binds to CD47 and prevents CD47 / SIRPa-mediated signaling, currently in Phase 1 trials (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein created by linking the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1, which works by binding to human CD47 and preventing it from delivering its "feeding" signal to macrophages, currently in Phase 1 clinical trials (NCT02890368 and NCT02663518); CC-90002 (Celgene), an anti-CD47 antibody used for leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc. is used for colorectal tumors and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0386] Checkpoint inhibitors that can be used in this invention include CD73 inhibitors. CD73 inhibitors currently being investigated in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody for solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody for solid tumors (NCT02754141).

[0387] Checkpoint inhibitors that can be used in this invention include agonists of interferon gene protein stimulators (STING, also known as transmembrane protein 173 or TMEM173). STING agonists being investigated in clinical trials include MK-1454 (Merck), an agonistic synthetic cyclic dinucleotide for lymphoma (NCT03010176); and ADU-S 100 (MIW815, Aduro Biotech / Novartis), an agonistic synthetic cyclic dinucleotide in Phase 1 trials (NCT02675439 and NCT03172936).

[0388] Checkpoint inhibitors that can be used in this invention include CSF1R inhibitors. CSF1R inhibitors currently being investigated in clinical trials include pericidatinib (PLX3397, Plexxikon), a small molecule CSF1R inhibitor used for colorectal cancer, pancreatic cancer, metastatic and advanced cancer (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 IMC-CS4 (LY3022855, Lilly). It is an anti-CSF-1R antibody used for pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid methylamide, Novartis), an orally available inhibitor of CSF1R used for advanced solid tumors (NCT02829723).

[0389] Checkpoint inhibitors that can be used in this invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors being investigated in clinical trials include monazolizumab (IPH2201, Innate Pharma), an anti-NKG2A antibody used for head and neck cancers (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0390] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pildizumab.

[0391] Example

[0392] The following examples are intended to illustrate the invention and should not be construed as limiting it. Unless otherwise stated, all amino acids are used in the L-configuration.

[0393]

[0394]

[0395] Example 1. Multitumor survey of Nectin-4 expression used to guide BT8009 indication selection

[0396] 1.1 Nectin-4 IHC Protocol

[0397] Reagents / Probes / Antibodies

[0398]

[0399] equipment

[0400]

[0401]

[0402] program

[0403] 1. Fix and embed the tissue, cut sections and mount them onto positively charged glass slides, and dewax and rehydrate the sections according to standard operating procedures.

[0404] 2. Load the sample into the Leica Bond III.

[0405] 3. Incubate with Bond dewaxing solution for 2 minutes at room temperature.

[0406] 4. Incubate with alcohol at room temperature for 2 minutes.

[0407] 5. Wash with Bond washing solution

[0408] 6. Incubate with Bond epitope repair solution 1 at 100°C for 20 minutes.

[0409] 7. Rinse with Bond washing solution.

[0410] 8. Seal with Bond peroxide sealant for 5 minutes at ambient temperature.

[0411] 9. Rinse with Bond washing solution.

[0412] 10. Block with Dako protein blocking agent at ambient temperature for 10 minutes.

[0413] 11. Rinse with Bond washing solution.

[0414] 12. Incubate with the anti-Nectin-4 antibody (10 μg / ml) diluted in Dako background de-diluent at ambient temperature for 30 minutes.

[0415] 13. Rinse with Bond washing solution.

[0416] 14. Incubate with the primary reagent at ambient temperature for 15 minutes.

[0417] 15. Rinse with Bond washing solution.

[0418] 16. Incubate with Bond polymer at ambient temperature for 15 minutes.

[0419] 17. Rinse with Bond washing solution.

[0420] 18. Incubate with Bond mixed with DAB purified reagent (Part 1) at ambient temperature, then incubate with Bond mixed with DAB purified reagent (Part 2) for 10 minutes.

[0421] 19. Rinse with DI water.

[0422] 20. Incubate with Bond hematoxylin for 5 minutes at ambient temperature.

[0423] 21. Rinse with DI water.

[0424] 22. Rinse with Bond washing solution.

[0425] 23. Rinse with DI water.

[0426] 24. Remove, dehydrate in a gradient of alcohols, and clarify in xylene at ambient temperature for 7 minutes.

[0427] 25. Sealing

[0428] Nectin-4 IHC score

[0429] Nectin-4 staining results were scored using the H-score method (defined as the sum of the products of cell percentage and their staining intensity on a 0-3 scale, where 0 is negative and 3 is strong staining). Independent H-scores for the cell membrane and cytoplasm were generated to distinguish the two compartments.

[0430] 1.2 Results

[0431] 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 a BOND polymer-refined assay kit. Tumor microenvironment (TMA) samples 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 cancer) were stained and Nectin-4 levels were manually scored to evaluate the tumor microenvironment in subsets of samples. Nectin-4 H scores (based on staining intensity x percentage of positive tumor cells on a 0-3 scale) were generated individually by pathologists for tumor cell membranes and cytoplasm. An H score ≥100 in either the tumor membrane or cytoplasm was considered positive.

[0432]

[0433] Of all the indications tested, greater Nectin-4 positivity was observed in the tumor cytoplasm compared to the tumor membrane. Breast cancer and bladder cancer showed the highest frequencies of Nectin-4 positivity. Subtype analysis of breast cancer identified enrichment of Nectin-4 expression in both hormone receptor-negative and human epidermal growth factor receptor 2-positive tumors.

[0434] Conclusion: The frequency of Nectin-4 expression in various tumor types, as measured by IHC, can guide clinical strategies for the BT8009 procedure.

[0435] Example 2. Molecular-based enrichment strategy for the Nectin-4-targeting bicyclic toxin conjugate BT8009

[0436] Materials: Tumor nuclei were obtained from US Biomax (Rockville, MD) and used for TMA construction, followed by IHC staining, 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: 2 each of ER+, PR+, and HER2+; and Nectin-4 protein expression controls: 2 spleen tissues and 2 skin tissues).

[0437] Nectin-4 protein expression: IHC was performed using the Nectin-4 IHC assay. Tumor membrane and cytoplasmic H scores were determined by pathologists.

[0438] 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. The samples were then run through DNA QC, whole exome library construction (including library preparation and hybridization capture), and whole exome sequence generation. All samples that passed DNA QC and sequence metric cutoffs were moved to DNA variant analysis.

[0439] DNA variant analysis: Somatic analysis of tumor-only tumors was performed on a cohort of normal tissues using the GATK4 MuTect2 (SNV / Indels) and GATK4CNV pipelines.

[0440] Whole-exome sequencing data preprocessing: A total of 100 copy number segment files were pre-annotated and formatted using Oncotator. The annotated copy number segment files specified genomic locations, copy number calls (diploid, amplified, or deleted), the average log2-transformed copy ratio, and genes located within regions of each contiguous chromosomal segment with homoploidy. These segment data were then expanded to gene-level data, where genes were assigned allele frequencies for their parental chromosomal segments. Genes with homoploidy, as indicated by their presence in multiple chromosomal segments, were excluded from the analysis.

[0441] The aim of this experiment was to identify molecular substitutes (i.e., somatic mutations or gene amplifications) for routinely measured tumor Nectin-4 protein expression, which could potentially increase patient screening frequency, yield, and likelihood of response to BT8009. Nectin-4 copy number was demonstrated to be associated with Nectin-4 mRNA expression in nine cancer indications (including breast, bladder, and lung cancer) in the TCGA. Furthermore, in >30 cancer indications in the TCGA, Nectin-4 copy number was highly correlated with SDHC copy number (the two genes are approximately 225 kb apart at 1q23), and SDHC was included in multiple commercially available NGS panels. In summary, these results indicate 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 numbers and Nectin-4 protein expression status via IHC.

[0442] Results: A high positive correlation was observed between SDHC and Nectin-4 copy number (R0). 2=0.93). Furthermore, all (N=22) TNBC tumors with Nectin-4 copy number ≥3 were identified as Nectin-4 positive and at or above the H score threshold required for inclusion in BT8009-100. Conversely, in the Nectin-4 / SDHC diploid tumor subgroup (n=30), both Nectin-4 positive and negative tumors were present. This suggests that the presence of SDHC / Nectin-4 amplification can increase the likelihood of identifying tumors expressing Nectin-4.

[0443] Conclusion: Pre-existing patient NGS data with SDHC amplification has been demonstrated to be useful for identifying patients with tumors exhibiting high Nectin-4 expression via IHC.

[0444] Example 3. MultiOmyx TM Multiplex immunofluorescence assay.

[0445] MultiOmyx was used in FFPE samples from head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), breast cancer, and bladder cancer. TM The technology evaluated the expression of a set 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. Each FFPE slide was provided to NeoGenomics pathologists for tissue annotation and selection. Tumor-related regions selected by the pathologists were used for staining and analysis. Staining was performed using a single 4µM FFPE slide. In each round of staining, antibodies labeled with two cyanine dyes (Cy3, Cy5) were paired together and recognized up to two biomarkers. The staining signal was then imaged, followed by new dye inactivation, enabling repeated rounds of staining. A proprietary deep learning-based workflow was applied to identify individual cells and classify all individual biomarkers, as well as to identify tissue and tumor regions for analysis. The results of individual cell and region classifications are combined to generate a co-expression profile and spatial distribution statistics of phenotypes of interest are calculated.

[0446] A MultiOmyx™ ultramultiplex immunofluorescence assay was developed to simultaneously quantify the presence of Nectin-4 and CD137-positive cells and to create in situ topographic maps of these cell types from 15 primary human tumor samples. (e.g.) Figure 10B(As shown). The frequency of Nectin-4 and CD137-positive cells detected in each tumor sample demonstrates a considerable infiltration of CD137+ immune cells in Nectin-4-positive NSCLC, HNSCC, and bladder cancer samples. Spatial distribution analysis further revealed the detection of CD137+ immune infiltration within the tumor stroma, with some of these penetrating deeply into the tumor core and most closely approximating Nectin-4-positive tumor cells (as shown). Figure 10C In deeper distributions, the CD137-expressing immune cells detected in all tumor types analyzed primarily included CD4+ and CD8+ T cells and CD68+ macrophages. Figure 10D While the role of CD137TICA may not be limited to T cells, these data suggest that at least CD137-carrying T cells may come into contact with tumor cells carrying Nectin-4. Therefore, these observations support the development of tumor-targeting CD137 agonists for the treatment of Nectin-4-positive human cancers.

[0447] While several embodiments of the invention have been described, it will be apparent that variations may be made to provide other embodiments utilizing the compounds and methods of the invention. Therefore, it should be understood that the scope of the invention is defined by this application and the claims, and not by the specific embodiments illustrated by the examples. sequence list <110> BicycleTx Limited <120> Bicyclic conjugates specific to NECTIN-4 and their uses <130> BIC-C-P3105PCT <150> 63 / 066,434 <151> 2020-08-17 <150> 63 / 134,202 <151> 2021-01-06 <150> 63 / 171,654 <151> 2021-04-07 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <220> <221> Xaa <222> (1)..(1) <223> Xaa is B - Ala <220> <221> Xaa <222> (2)..(2) <223> Xaa is Sar10 <220> <221> Xaa <222> (5)..(5) <223> Xaa is 1Nal <220> <221> Xaa <222> (9)..(9) <223> Xaa is HArg <220> <221> Xaa <222> (15)..(15) <223> Xaa is HyP <400> 1 Xaa Xaa Cys Pro Xaa Asp Cys Met Xaa Asp Trp Ser Thr Pro Xaa Trp 1 5 10 15 Cys

Claims

1. Use in the preparation of a medicament for measuring the copy number of SDHC DNA in a patient’s tumor tissue or circulating tumor DNA (ctDNA), said medicament for identifying or selecting patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue.

2. The use as claimed in claim 1, wherein the step of measuring the SDHC DNA copy number in a patient’s tumor tissue or circulating tumor DNA (ctDNA) includes using next-generation sequencing (NGS) technology or array-based sequence capture.

3. The use in the preparation of a medicament for measuring the SDHC DNA copy number of a patient using next-generation sequencing (NGS) technology or array-based sequence capture, the medicament being used to identify or select patients with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, wherein the medicament for measuring the SDHC DNA copy number of a patient is used with next-generation sequencing (NGS) technology or array-based sequence capture, and patients with elevated SDHC DNA copy numbers are selected.

4. Use in the preparation of a medicament for measuring the copy number of DDR2 DNA in a patient’s tumor tissue or circulating tumor DNA (ctDNA), said medicament being used to identify or select patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue.

5. The use as claimed in claim 4, wherein the step of measuring the DDR2 DNA copy number in the patient's tumor tissue or circulating tumor DNA (ctDNA) includes using next-generation sequencing (NGS) technology or array-based sequence capture.

6. Use in the preparation of a medicament for measuring the DDR2 DNA copy number of a patient using next-generation sequencing (NGS) technology or array-based sequence capture, the medicament being used to identify or select patients with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue, wherein the medicament for measuring the DDR2 DNA copy number of a patient is used with next-generation sequencing (NGS) technology or array-based sequence capture, and patients with elevated DDR2 DNA copy numbers are selected.

7. The use as described in any one of claims 1-6, 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.

8. The use as described in any one of claims 1-3, 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.

9. The use as described in any one of claims 4-6, wherein the selected patient has a DDR2 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.

10. Use of a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating cancer in patients with elevated levels of Nectin-4 protein and / or RNA expression in tumor tissue. Patients with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue are defined as those with an SDHC Log2 (CN ratio) of approximately 0.1 or greater, approximately 0.2 or greater, approximately 0.3 or greater, approximately 0.4 or greater, approximately 0.5 or greater, approximately 0.6 or greater, approximately 0.7 or greater, approximately 0.8 or greater, approximately 0.9 or greater, or approximately 1.0 or greater, as measured in their tumor tissue or circulating tumor DNA (ctDNA). Patients with elevated Nectin-4 protein and / or RNA expression levels in tumor tissue are defined as those with a DDR2 Log2 (CN ratio) of approximately 0.1 or greater, approximately 0.2 or greater, approximately 0.3 or greater, approximately 0.4 or greater, approximately 0.5 or greater, approximately 0.6 or greater, approximately 0.7 or greater, approximately 0.8 or greater, approximately 0.9 or greater, or approximately 1.0 or greater, as measured in the tumor tissue or circulating tumor DNA (ctDNA).

11. The use of a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in the preparation of a medicament for treating cancer in patients, including the selection of patients whose SDHC Log2 (CN ratio) in tumor tissue or in circulating tumor DNA (ctDNA) is 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, as determined by next-generation sequencing (NGS) technology or array-based sequence capture.

12. The use of a bicyclic toxin conjugate or bicyclic TICA specific for Nectin-4 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in the preparation of a medicament for treating cancer in patients, including the selection of patients whose DDR2 Log2 (CN ratio) in tumor tissue or circulating tumor DNA (ctDNA) is 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, as determined by next-generation sequencing (NGS) technology or array-based sequence capture.

13. The use as described in any one of claims 10-12, 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.

14. The method further comprises administering an immunotumor agent in any one of claims 10-12.

15. The use as described in claim 14, wherein the immunotumor agent is a PD-1 antagonist.

16. The use as described in claim 15, wherein the PD-1 antagonist is an antagonistic PD-1 antibody.

17. The use as described in claim 16, wherein the antagonistic PD-1 antibody is selected from nivolumab, pembrolizumab, MEDI-0680, pildizumab, AMP-224, atezolizumab, durvalumab, BMS-936559, avelumab, or cimiprimab.

18. The use as claimed in any one of claims 10-12, wherein the bicyclic toxin conjugate specific for Nectin-4 is BT8009 or the bicyclic TICA is BT7480.

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