BIOMARKERS FOR CBL, AND COMPOSITIONS AND METHODS FOR THEIR USE

FIELD

Provided herein are biomarkers for CBL inhibition, methods of their use for measuring CBL activity in vivo, and compositions and methods for their use in therapy.

BACKGROUND

Casitas B-lineage lymphoma proto-oncogene (CBL) has been characterized as a regulator of the adaptive immune response (Bachmaier et al., 2000, Nature 403(6766):211-6; Chiang et al., 2000, Nature 403(6766):216-20). CBL functions as an intracellular checkpoint that regulates T cell activation, NK cell activity, and immune response through degradation of specific proteins. Both T cell receptor (TCR) signaling and B cell receptor (BCR) signaling are modulated by CBL mediated ubiquitination. Recent studies have identified CBL inhibition as a promising target for therapy. Inhibition of CBL with a small molecule drug activates T cells, a goal in the treatment of cancer, where T cells and the entire immune system can be mobilized to destroy a tumor. However, detection and measurement of CBL inhibition in vivo can be challenging. Practical in vivo markers for CBL are needed for the study and use of these promising CBL therapeutics.

SUMMARY

The methods and compositions provided herein are based, in part, on the discovery of proximal biomarkers of CBL inhibition that show statistically significant correlation with activity in vivo. As demonstrated in the examples provided herein, statistically significant increases of phosphorylated interleukin-2-inducible kinase (ITK), phosphorylated hematopoietic lineage cell-specific protein (HS1), phosphorylated phospholipase C2 (PLCγ2), phosphorylated phospholipase C1 (PLCγ1), phosphorylated spleen tyrosine kinase (SYK), and phosphorylated Zeta-chain-associated protein kinase 70 (ZAP70) correlated with CBL inhibition in vitro and in vivo. ITK is a T-cell-specific kinase inducible by interleukin 2 (Siliciano et al., 1992, Proc Natl Acad Sci USA 89(23):11194-8). HS1 binds the actin cytoskeleton and regulates TCR activation through the immune synapse (Yamanashi et al., 1993, Proc. Natl. Acad. Sci. USA 90(8):3631-5; Scaife et al., 2003, J. Cell Sci. 116(3): 463-473; Gomez et al., 2006, Immunity 24:741-752; Carrizosa et al., 2009, J. Immunol. 183(11):7352-61). SYK is a tyrosine kinase involved in TCR signaling (Chan et al., 1994, J. Immunol. 152(10):4758-4766). PLCγ2, PLCγ1, and ZAP70 are expressed in T cells and activated upon TCR stimulation. (Rhee et al., 2001, Annual Review of Biochemistry. 70:281-312; Fu et al., 2010, J. Experimental Med. 207(2):309-318; Wang et al., 2010, Cold Spring Harbor Persp. Biol. 2(5):a002279).

In one aspect, provided herein are methods of measuring proximal biomarkers of CBL inhibition in a sample. In certain embodiments, the methods comprise the steps of measuring the amount of a proximal biomarker of CBL inhibition in the sample. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70.

In one aspect, provided herein are methods for detecting the amount of biomarkers of CBL inhibition in a cell, tissue, or organism. In certain embodiments, the methods comprise the steps of measuring the amount of a proximal biomarker of CBL inhibition in a first sample of the cell, tissue, or organism; measuring the amount of a proximal biomarker of CBL inhibition in a second sample of the cell, tissue, or organism; and identifying a change in the amount of CBL in the cell, tissue, or organism based on the change in the amount of the biomarker detected in the sample. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70.

In one aspect, provided herein are methods of treating a patient in need thereof with a CBL inhibitor. In certain embodiments, the methods comprise administering to a patient a first dose of a CBL inhibitor; measuring the amount of a CBL biomarker in a first sample of the patient; and identifying the amount of CBL in the patient based on the amount of the CBL biomarker detected. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70.

In one aspect, provided herein are methods of treating a patient in need thereof with a CBL inhibitor. In certain embodiments, the methods comprise administering to a patient a first dose of a CBL inhibitor; measuring the amount of a CBL biomarker in a first sample of the cell, tissue, or organism; measuring the amount of a CBL biomarker in a second sample of the cell, tissue, or organism; and identifying a change in the amount of CBL in the cell, tissue, or organism based on the change in the amount of the CBL biomarker detected in the sample. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70.

In another aspect, provided herein are compositions and kits for carrying out the methods. The compositions and methods are useful for treating or preventing any disease or disorder modulated by CBL. In certain embodiments, the compositions and methods are useful for treating a proliferative disorder. In certain embodiments, the CBL inhibitor is a small molecule. In certain embodiments, CBL inhibitor is a small molecule administered in combination with a cell therapy. Useful CBL inhibitors and therapies are described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A provides single dose Compound 23 in cynomolgus monkeys induces phosphorylated PLCγ2 upon stimulation of circulating CD8⁺ T cells.

FIG. 1B provides single dose Compound 23 in cynomolgus monkeys induces phosphorylated HS1 upon stimulation of circulating CD8⁺ T cells.

FIG. 1C provides single dose Compound 23 in cynomolgus monkeys induces phosphorylated HS1 upon stimulation of circulating CD8⁺ T cells.

FIG. 2A provides multiple doses of Compound 23 in cynomolgus monkeys induces phosphorylated PLCγ2 upon stimulation of circulating CD8⁺ T cells.

FIG. 2B provides multiple doses of Compound 23 in cynomolgus monkeys induces phosphorylated HS1 upon stimulation of circulating CD8⁺ T cells.

FIG. 2C provides multiple doses of Compound 23 in cynomolgus monkeys induces phosphorylated ZAP70 upon stimulation of circulating CD8⁺ T cells.

FIG. 3A provides Compound 23 induces phosphorylated HS1 upon stimulation in vitro in human CD8⁺ T cells from whole blood.

FIG. 3B provides Compound 23 induces phosphorylated PLCγ2 upon stimulation in vitro in human CD8⁺ T cells from whole blood.

FIG. 3C provides Compound 23 induces phosphorylated ZAP70 upon stimulation in vitro in human CD8⁺ T cells from whole blood.

FIG. 4A provides Compound 23 induces phosphorylated HS1 upon stimulation in vitro in cynomolgus macaque CD8⁺ T cells from whole blood.

FIG. 4B provides Compound 23 induces phosphorylated PLCγ2 upon stimulation in vitro in cynomolgus macaque CD8⁺ T cells from whole blood.

FIG. 4C provides Compound 23 induces phosphorylated ZAP70 upon stimulation in vitro in cynomolgus macaque CD8⁺ T cells from whole blood.

FIG. 5A provides an illustration of methods for identifying proximal biomarkers of Compound 23 described herein.

FIG. 5B provides results of the methods, identifying markers pHS1, PLCγ2, and pZAP70.

FIG. 5C provides results of the methods, identifying markers pITK, pHS1, PLCγ1, PLCγ2, pSYK, and pZAP70.

FIG. 6A provides dose dependent activation of Compound 23 proximal biomarkers pHS1, PLCγ2, and pZAP70 with a 5-minute stimulation.

FIG. 6B provides dose dependent activation of Compound 23 proximal biomarkers pHS1, PLCγ2, and pZAP70 with a 60-minute stimulation.

FIG. 7 provides Compound 23 eliciting a concentration-dependent increase in phosphorylated HS1 in human whole blood.

FIG. 8A provides dose-dependent phosphorylation of HS1 on administration of Compound 23 to human patients.

FIG. 8B provides maximal percent HS1 phosphorylation on administration of Compound 23 to human patients.

FIG. 8C provides area under the curve percent phosphorylated HS1 on administration of Compound 23 to human patients.

FIG. 9A provides maximal percent PLCγ2 phosphorylation on administration of Compound 23 to human patients.

FIG. 9B provides area under the curve percent phosphorylated PLCγ2 on administration of Compound 23 to human patients.

FIG. 10A provides maximal percent ZAP70 phosphorylation on administration of Compound 23 to human patients.

FIG. 10B provides area under the curve percent phosphorylated ZAP70 on administration of Compound 23 to human patients.

DETAILED DESCRIPTION

Provided herein are biomarkers, compositions, and methods useful for treating proliferative disorders in a subject.

1. Definitions

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless otherwise indicated or clear from context. For example, “an” excipient includes one or more excipients.

Reference to “about” a value, encompasses from 90% to 110% of that value. For instance, about 50 billion cells refers to 45 to 55 billion cells, and includes 50 billion cells. For instance, a temperature of “about 100 degrees” refers to a temperature of about 90 degrees to about 110 degrees.

When numerical ranges of compounds are given, all compounds within those numerical limits designated “a” and “b” are included, unless expressly excluded. For example, reference to compounds 9-13 refers to compounds 9, 10, 11, 12, and 13.

The term “CBL” refers to any Casitas B-lineage lymphoma gene encoding one of the CBL family of proteins. CBL family proteins include c-Cbl and Cbl-b.

The term “Cbl-b” as used herein refers to a Cbl-b protein. The term also includes naturally occurring variants of Cbl-b, including splice variants or allelic variants. The term also includes non-naturally occurring variants of Cbl-b, such as a recombinant Cbl-b protein or truncated variants thereof, which generally preserve the binding ability of naturally occurring Cbl-b or naturally occurring variants of Cbl-b (e.g., the ability to bind to an E2 enzyme). Sequences include NM_170662, NM_001321786, NM_001321788, NM_001321789, NM_001321790, and NM_001033238 (mRNA); and NP_001308715, NP_001308717, NP_001308718, NP_001308719, and NP_001308720 (protein).

The term “c-Cbl” as used herein refers to a c-Cbl protein. The term also includes naturally occurring variants of c-Cbl, including splice variants or allelic variants. The term also includes non-naturally occurring variants of c-Cbl, such as a recombinant c-Cbl protein or truncated variants thereof, which generally preserve the binding ability of naturally occurring c-Cbl or naturally occurring variants of c-Cbl (e.g., the ability to bind to an E2 enzyme). Sequences include NM_005188 human and NM_007619 mouse(mRNA); and NP_005179 human and NP_013645 mouse (protein).

The term “ITK” as used herein refers to a interleukin-2-inducible T-cell kinase. In humans, ITK is encoded by the ITK gene. The term “pITK” refers to phosphorylated ITK. The term also includes naturally occurring variants of ITK, including splice variants or allelic variants. Sequences include NM_005546 (mRNA); and NP_005537 (protein). In certain embodiments, pITK is phosphorylated on residue Y180.

The term “HS1” as used herein refers to a hematopoietic lineage cell-specific protein. In humans, HS1 is encoded by the HCLS1 gene. The term “pHS1” refers to phosphorylated HS1. The term also includes naturally occurring variants of HS1, including splice variants or allelic variants. Sequences include NM_005335 and NM_001292041 (mRNA); and NP_005326 and NP_0001278970 (protein). In certain embodiments, pHS1 is phosphorylated on residue Y397.

The term “PLCγ2” as used herein refers to a phosphatidylinositol-specific phospholipase C, gamma 2 protein. In humans, pPLCγ2 is encoded by the PLCG2 gene. The term “pPLCγ2” refers to phosphorylated PLCγ2. The term also includes naturally occurring variants of PLCγ2, including splice variants or allelic variants. Sequences include NM_002661 (mRNA); and NP_002652 (protein). In certain embodiments, PLCγ2 is phosphorylated on residue Y759.

The term “PLCγ1” as used herein refers to a phosphatidylinositol-specific phospholipase C, gamma 1 protein. In humans, pPLCγ1 is encoded by the PLCG1 gene. The term “pPLCγ1” refers to phosphorylated PLCγ1. The term also includes naturally occurring variants of PLCγ1, including splice variants or allelic variants. Sequences include NM_002660 and NM_182811 (mRNA); and NP_002651 and NP_877963 (protein). In certain embodiments, PLCγ1 is phosphorylated on residue Y783.

The term “SYK” as used herein refers to a tyrosine-protein kinase, or spleen tyrosine kinase, protein. In humans, SYK is encoded by the SYK gene. The term “pSYK” refers to phosphorylated SYK. The term also includes naturally occurring variants of SYK, including splice variants or allelic variants. Sequences include NM_001135052, NM_001174167, NM_001174168, and NM_003177 (mRNA); and NP_001128524, NP_001167638, NP_001167639, and, NP_003168 (protein). In certain embodiments, pSYK is phosphorylated on residue Y525, Y526, and/or Y348.

The term “ZAP70” as used herein refers to a zeta-chain-associated protein kinase 70 protein. In humans, ZAP70 is encoded by the ZAP70 gene. The term “pZAP70” refers to phosphorylated ZAP70. The term also includes naturally occurring variants of ZAP70, including splice variants or allelic variants. Sequences include NM_001079, NM_207519, and NM_001378594 (mRNA); and NP_001070, NP_997402, NP_001365523 (protein). In certain embodiments, pZAP70 is phosphorylated on residue Y319.

The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to preparations that are in such form as to permit the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to an individual to which the formulation or composition would be administered. Such formulations or compositions may be sterile. Such formulations or compositions may be sterile, with the exception of the inclusion of an oncolytic virus.

“Excipients” as used herein include pharmaceutically acceptable excipients, carriers, vehicles, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable excipient is an aqueous pH buffered solution.

Reference to a compound as described in a pharmaceutical composition, or to a compound as described in a claim to a pharmaceutical composition, refers to the compound described by the formula recited in the pharmaceutical composition, without the other elements of the pharmaceutical composition, that is, without carriers, excipients, etc.

“Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder.

As used herein, the term “inhibits growth” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in cell growth (e.g., tumor cell growth) when contacted with a compound or combination described herein, as compared to the growth of the same cells not in contact with the same compound or combination. In certain embodiments, growth may be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. The decrease in cell growth can occur by a variety of mechanisms, including but not limited to antibody internalization, apoptosis, necrosis, and/or effector function-mediated activity.

As used herein, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, avians, goats, and sheep. In certain embodiments, the subject is a human. In certain embodiments, the subject has a disease that can be treated or diagnosed with a CBL inhibitor provided herein. In certain embodiments, the disease is gastric carcinoma, colorectal carcinoma, renal cell carcinoma, cervical carcinoma, non-small cell lung carcinoma, ovarian cancer, breast cancer, triple-negative breast cancer, endometrial cancer, prostate cancer, and/or a cancer of epithelial origin.

An “effective amount” of an agent disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose. An “effective amount” or an “amount sufficient” of an agent is that amount adequate to produce a desired biological effect, such as a beneficial result, including a beneficial clinical result. In certain embodiments, the term “effective amount” refers to an amount of an agent effective to “treat” a disease or disorder in an individual (e.g., a mammal such as a human).

“Proliferation” is used herein to refer to the proliferation of a cell. Increased proliferation encompasses the production of a greater number of cells relative to a baseline value. Decreased proliferation encompasses the production of a reduced number of cells relative to a baseline value. In certain embodiments, the cell is an immune cell such as a T-cell and increased proliferation is desired. In certain embodiments, the cell is a cancer cell and reduced proliferation is desired.

“Alkyl” as used herein refers to a saturated linear (i.e., unbranched) or branched univalent hydrocarbon chain or combination thereof. Particular alkyl groups are those having a designated number of carbon atoms, for example, an alkyl group having 1 to 20 carbon atoms (a “C₁-C₂₀alkyl”), having 1 to 10 carbon atoms (a “C₁-C₁₀” alkyl), having 1 to 8 carbon atoms (a “C₁-C₈alkyl”), having 1 to 6 carbon atoms (a “C₁-C₆alkyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkyl”), or having 1 to 4 carbon atoms (a “C₁-C₄alkyl”). Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

“Alkenyl” as used herein refers to an unsaturated linear (i.e., unbranched) or branched univalent hydrocarbon chain or combination thereof, having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C═C). Particular alkenyl groups are those having a designated number of carbon atoms, for example, an alkenyl group having 2 to 20 carbon atoms (a “C₂-C₂₀alkenyl”), having 2 to 10 carbon atoms (a “C₂-C₁₀” alkenyl), having 2 to 8 carbon atoms (a “C₂-C₈alkenyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkenyl”), or having 2 to 4 carbon atoms (a “C₂-C₄alkenyl”). The alkenyl group may be in “cis” or “trans” configurations or, alternatively, in “E” or “Z” configurations. Examples of alkenyl groups include, but are not limited to, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs and isomers thereof, and the like.

“Alkynyl” as used herein refers to an unsaturated linear (i.e., unbranched) or branched univalent hydrocarbon chain or combination thereof, having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). Particular alkynyl groups are those having a designated number of carbon atoms, for example, an alkynyl group having 2 to 20 carbon atoms (a “C₂-C₂₀alkynyl”), having 2 to 10 carbon atoms (a “C₂-C₁₀alkynyl”), having 2 to 8 carbon atoms (a “C₂-C₈alkynyl”), having 2 to 6 carbon atoms (a “C₂-C₆alkynyl”), or having 2 to 4 carbon atoms (a “C₂-C₄alkynyl”). Examples of alkynyl groups include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs, and isomers thereof, and the like.

“Alkylene” as used herein refers to the same residues as alkyl, but having bivalency. Particular alkylene groups are those having 1 to 6 carbon atoms (a “C₁-C₆alkylene”), 1 to 5 carbon atoms (a “C₁-C₅alkylene”), 1 to 4 carbon atoms (a “C₁-C₄alkylene”), or 1 to 3 carbon atoms (a “C₁-C₃alkylene”). Examples of alkylene groups include, but are not limited to, groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), butylene (—CH₂CH₂CH₂CH₂—), and the like.

“Cycloalkyl” as used herein refers to non-aromatic, saturated or unsaturated, cyclic univalent hydrocarbon structures. Particular cycloalkyl groups are those having a designated number of annular (i.e., ring) carbon atoms, for example, a cycloalkyl group having from 3 to 12 annular carbon atoms (a “C₃-C₁₂cycloalkyl”). A particular cycloalkyl is a cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a “C₃-C₈cycloalkyl”), or having 3 to 6 annular carbon atoms (a “C₃-C₆cycloalkyl”). Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl, but excludes aryl (i.e., aromatic) groups. A cycloalkyl comprising more than one ring may be fused, spiro, or bridged, or combinations thereof. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl

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cyclobutyl

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cyclopentyl

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cyclohexyl

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1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl

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norbornyl, and the like.

“Cycloalkylene” as used herein refers to the same residues as cycloalkyl, but having bivalency. Particular cycloalkylene groups are those having 3 to 12 annular carbon atoms (a “C₃-C₁₂cycloalkylene”), having from 3 to 8 annular carbon atoms (a “C₃-C₈cycloalkylene”), or having 3 to 6 annular carbon atoms (a “C₃-C₆cycloalkylene”). Examples of cycloalkylene groups include, but are not limited to, cyclopropylene

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cyclopentylene

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cyclopentylene

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cyclohexylene

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1,2-cyclohexenylene, 1,3-cyclohexenylene, 1,4-cyclohexenylene, cycloheptylene

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norbornylene, and the like.

“Aryl” as used herein refers to an aromatic carbocyclic group having a single ring (e.g., phenyl), or multiple condensed rings (e.g., naphthyl or anthryl) where one or more of the condensed rings may not be aromatic. Particular aryl groups are those having from 6 to 14 annular (i.e., ring) carbon atoms (a “C₆-C₁₄aryl”). An aryl group having more than one ring where at least one ring is non-aromatic may be connected to the parent structure at either an aromatic ring position or at a non-aromatic ring position. In one variation, an aryl group having more than one ring where at least one ring is non-aromatic is connected to the parent structure at an aromatic ring position. Examples of aryls include, but are not limited to, groups such as phenyl, naphthyl, 1-naphthyl, 2-naphthyl, 1,2,3,4-tetrahydronaphthalen-6-yl

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and the like.

“Carbocyclyl” or “carbocyclic” refers to an aromatic or non-aromatic univalent cyclic group in which all of the ring members are carbon atoms, such as cyclohexyl, phenyl, 1,2-dihydronaphthyl, etc.

“Arylene” as used herein refers to the same residues as aryl, but having bivalency. Particular arylene groups are those having from 6 to 14 annular carbon atoms (a “C₆-C₁₄arylene”). Examples of arylene include, but are not limited to, groups such as phenylene, o-phenylene (i.e., 1,2-phenylene), m-phenylene (i.e., 1,3-phenylene), p-phenylene (i.e., 1,4-phenylene), naphthylene, 1,2-naphthylene, 1,3-naphthylene, 1,4-naphthylene, 2,7-naphthylene, 2,6-naphthylene, and the like.

“Heteroaryl” as used herein refers to an unsaturated aromatic cyclic group having from 1 to 14 annular carbon atoms and at least one annular heteroatom, including, but not limited to, heteroatoms such as nitrogen (N), oxygen (O), and sulfur (S). A heteroaryl group may have a single ring (e.g., pyridyl or imidazolyl) or multiple condensed rings (e.g., indolizinyl, indolyl, or quinolinyl) where at least one of the condensed rings is aromatic. Particular heteroaryl groups are 5- to 14-membered rings having 1 to 12 annular carbon atoms and 1 to 6 annular heteroatoms independently selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S) (a “5- to 14-membered heteroaryl”); 5- to 10-membered rings having 1 to 8 annular carbon atoms and 1 to 4 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (a “5- to 10-membered heteroaryl”); or 5-, 6-, or 7-membered rings having 1 to 5 annular carbon atoms and 1 to 4 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (a “5- to 7-membered heteroaryl”). In one variation, heteroaryl includes monocyclic aromatic 5-, 6-, or 7-membered rings having from 1 to 6 annular carbon atoms and 1 to 4 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In another variation, heteroaryl includes polycyclic aromatic rings having from 1 to 12 annular carbon atoms and 1 to 6 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. A heteroaryl group having more than one ring where at least one ring is non-aromatic may be connected to the parent structure at either an aromatic ring position or at a non-aromatic ring position. Examples of heteroaryl include, but are not limited to, groups such as pyridyl, benzimidazolyl, benzotriazolyl, benzo[b]thienyl, quinolinyl, indolyl, benzothiazolyl, and the like. “Heteroaryl” also includes moieties such as

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(2,4-dihydro-3H-1,2,4-triazol-3-one-2-yl), which has the aromatic tautomeric structure

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(1H-1,2,4-triazol-5-ol-1-yl).

“Heterocyclyl” and “heterocyclic groups” as used herein refer to non-aromatic saturated or partially unsaturated cyclic groups having the number of atoms and heteroatoms as specified, or if no number of atoms or heteroatoms is specified, having at least three annular atoms, from 1 to 14 annular carbon atoms, and at least one annular heteroatom, including, but not limited to, heteroatoms such as nitrogen, oxygen, and sulfur. A heterocyclic group may have a single ring (e.g., tetrahydrothiophenyl, oxazolidinyl) or multiple condensed rings (e.g., decahydroquinolinyl, octahydrobenzo[d]oxazolyl). Multiple condensed rings include, but are not limited to, bicyclic, tricyclic, and quadracylic rings, as well as bridged or spirocyclic ring systems. Examples of heterocyclic groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxazolidinyl, piperazinyl, morpholinyl, dioxanyl, 3,6-dihydro-2H-pyranyl, 2,3-dihydro-1H-imidazolyl, and the like.

“Heteroarylene” as used herein refers to the same residues as heteroaryl, but having bivalency. Particular heteroarylene groups are 5- to 14-membered rings having 1 to 12 annular carbon atoms and 1 to 6 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (a “5- to 14-membered heteroarylene”); 5- to 10-membered rings having 1 to 8 annular carbon atoms and 1 to 4 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (a “5- to 10-membered heteroarylene”); or 5-, 6-, or 7-membered rings having 1 to 5 annular carbon atoms and 1 to 4 annular heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (a “5- to 7-membered heteroarylene”). Examples of heteroarylene include, but are not limited to, groups such as pyridylene, benzimidazolylene, benzotriazolylene, benzo[b]thienylene, quinolinylene, indolylene, benzothiazolylene, and the like.

“Halo” or “halogen” refers to elements of the Group 17 series having atomic number 9 to 85. Halo groups include fluoro (F), chloro (Cl), bromo (Br), and iodo (I).

“Haloalkyl,” “haloalkylene,” “haloaryl,” “haloarylene,” “haloheteroaryl,” and similar terms refer to a moiety substituted with at least one halo group. Where a haloalkyl moiety or other halo-substituted moiety is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached. For example, dihaloaryl, dihaloalkyl, trihaloaryl, trihaloalkyl, etc., refer to aryl and alkyl substituted with two (“di”) or three (“tri”) halo groups, which may be, but are not necessarily, the same halo; thus, for example, the haloaryl group 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. The subset of haloalkyl groups in which each hydrogen (H) of an alkyl group is replaced with a halo group is referred to as a “perhaloalkyl.” A particular perhaloalkyl group is trifluoroalkyl (—CF₃). Similarly, “perhaloalkoxy” refers to an alkoxy group in which a halogen takes the place of each hydrogen (H) in the hydrocarbon making up the alkyl moiety of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (—OCF₃). “Haloalkyl” includes monohaloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl, and any other number of halo substituents possible on an alkyl group; and similarly for other groups such as haloalkylene, haloaryl, haloarylene, haloheteroaryl, etc.

“Amino” refers to the group —NH₂.

“Oxo” refers to the group ═O, that is, an oxygen atom doubly bonded to carbon or another chemical element.

“Optionally substituted,” unless otherwise specified, means that a group is unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4, or 5) of the substituents listed for that group, in which the substituents may be the same or different. In one embodiment, an optionally substituted group is unsubstituted. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In certain embodiments, an optionally substituted group has 1 to 2, 1 to 3, 1 to 4, or 1 to 5 substituents. When multiple substituents are present, each substituent is independently chosen unless indicated otherwise. For example, each (C₁-C₄alkyl) substituent on the group —N(C₁-C₄alkyl)(C₁-C₄alkyl) can be selected independently from the other, so as to generate groups such as —N(CH₃)(CH₂CH₃), etc.

In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms (H) of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined herein. In certain embodiments, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or one substituent.

Substituents can be attached to any chemically possible location on the specified group or radical, unless indicated otherwise. Thus, in one embodiment, —C₁-C₈alkyl-OH includes, for example, —CH₂CH₂OH, —CH(OH)—CH₃, —CH₂C(OH)(CH₃)₂, and the like. By way of further example, in one embodiment, —C₁-C₆alkyl-OH includes, for example, —CH₂CH₂OH, —CH(OH)—CH₃, —CH₂C(OH)(CH₃)₂, and the like. By way of further example, in one embodiment, —C₁-C₆alkyl-CN includes, for example, —CH₂CH₂CN, —CH(CN)—CH₃, —CH₂C(CN)(CH₃)₂, and the like.

Unless a specific isotope of an element is indicated in a formula, the disclosure includes all isotopologues of the compounds disclosed herein, such as, for example, deuterated derivatives of the compounds (where H can be 2H, i.e., deuterium (D)). Deuterated compounds may provide favorable changes in pharmacokinetic (ADME) properties. Isotopologues can have isotopic replacements at any or at all locations in a structure, or can have atoms present in natural abundance at any or all locations in a structure.

A “small molecule” as used herein refers to a compound of 1,000 daltons or less in molecular weight.

Hydrogen atoms can also be replaced with close bioisosteres, such as fluorine, provided that such replacements result in stable compounds.

The disclosure also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the compounds described herein, and cis/trans or E/Z isomers. Unless stereochemistry is explicitly indicated in a chemical structure or name, the structure or name is intended to embrace all possible stereoisomers of a compound depicted. In addition, where a specific stereochemical form is depicted, it is understood that all other stereochemical forms are also described and embraced by the disclosure, as well as the general non-stereospecific form and mixtures of the disclosed compounds in any ratio, including mixtures of two or more stereochemical forms of a disclosed compound in any ratio, such that racemic, non-racemic, enantioenriched, and scalemic mixtures of a compound are embraced. Compositions comprising a disclosed compound also are intended, such as a composition of a substantially pure compound, including a specific stereochemical form thereof. Compositions comprising a mixture of disclosed compounds in any ratio also are embraced by the disclosure, including compositions comprising mixtures of two or more stereochemical forms of a disclosed compound in any ratio, such that racemic, non-racemic, enantioenriched, and scalemic mixtures of a compound are embraced by the disclosure. If stereochemistry is explicitly indicated for one portion or portions of a molecule, but not for another portion or portions of a molecule, the structure is intended to embrace all possible stereoisomers for the portion or portions where stereochemistry is not explicitly indicated.

This disclosure also embraces any and all tautomeric forms of the compounds described herein.

The disclosure is intended to embrace all salts of the compounds described herein, as well as methods of using such salts of the compounds. In one embodiment, the salts of the compounds comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that can be administered as drugs or pharmaceuticals to humans and/or animals and that, upon administration, retain at least some of the biological activity of the free compound (i.e., neutral compound or non-salt compound). The desired salt of a basic compound may be prepared by methods known to those of skill in the art by treating the compound with an acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids, and salicylic acid. Salts of basic compounds with amino acids, such as aspartate salts and glutamate salts, also can be prepared. The desired salt of an acidic compound can be prepared by methods known to those of skill in the art by treating the compound with a base. Examples of inorganic salts of acid compounds include, but are not limited to, alkali metal and alkaline earth salts, such as sodium salts, potassium salts, magnesium salts, and calcium salts; ammonium salts; and aluminum salts. Examples of organic salts of acid compounds include, but are not limited to, procaine, dibenzylamine, N-ethylpiperidine, N,N′-dibenzylethylenediamine, and triethylamine salts. Salts of acidic compounds with amino acids, such as lysine salts, also can be prepared. For lists of pharmaceutically acceptable salts, see, for example, P. H. Stahl and C. G. Wermuth (eds.) “Handbook of Pharmaceutical Salts, Properties, Selection and Use” Wiley-VCH, 2011 (ISBN: 978-3-906-39051-2). Several pharmaceutically acceptable salts are also disclosed in Berge, J. Pharm. Sci. 66(1):1-19 (1977).

2. CBL Biomarkers

In one aspect, provided herein are methods of detecting proximal biomarkers of CBL inhibition in a sample. In certain embodiments, embodiments, the methods comprise the steps of measuring the amount of a proximal biomarker of CBL inhibition in the sample.

The CBL biomarker can be any CBL biomarker deemed suitable by the person of skill. In certain embodiments, the CBL biomarker is a biomarker that correlates with CBL levels in vivo. In certain embodiments, the CBL biomarker is a biomarker that correlates with CBL expression in vivo. In certain embodiments, the CBL biomarker is a biomarker that correlates with CBL activity in vivo. In certain embodiments, the CBL biomarker is a biomarker that correlates with CBL protein levels in vivo. In certain embodiments the CBL biomarker is directly proportional to CBL level in vivo. In certain embodiments, the CBL biomarker is a biomarker that is directly proportional to CBL level in vivo to within a useful error of measurement.

In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is two of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is three of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is four of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is five of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is six of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70. In certain embodiments, the biomarker is pHS1, PLCγ2, and pZAP70. In other words, in certain embodiments, pHS1, pPLCγ2, and pZAP70 are measured to assess CBL level in vivo.

In certain embodiments, the methods detect CBL level in the sample. In certain embodiments, the methods detect CBL expression in the sample. In certain embodiments, the methods detect CBL activity in the sample. In certain embodiments, the methods detect CBL protein amount in the sample. In certain embodiments, the methods detect inhibition of CBL level in the sample. In certain embodiments, the methods detect inhibition of CBL expression in the sample. In certain embodiments, the methods detect inhibition of CBL activity in the sample. In certain embodiments, the methods detect inhibition of CBL protein amount in the sample. In certain embodiments, the methods detect degradation of CBL in the sample.

In certain embodiments, the sample is from a mammal. In certain embodiments, the sample is from a non-human mammal. In certain embodiments, the sample is from a human. In certain embodiments, the sample is from a human in need of treatment for a disease or disorder treatable with a CBL inhibitor. In certain embodiments, the sample is a tissue sample. In certain embodiments, the sample is a cell sample. In certain embodiments, the sample is a blood sample. In certain embodiments, the sample is a plasma sample. In certain embodiments, the sample is a tumor sample. In certain embodiments, the sample is a tumor biopsy. In certain embodiments, the sample is a cell culture.

The CBL can be any CBL deemed suitable to the person of skill. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In other words, in certain embodiments, the biomarker is useful for assessing Cbl-b and c-Cbl levels in vivo.

In another aspect, provided herein are methods for detecting the amount of CBL in a cell, tissue, or organism. In certain embodiments, the methods comprise the steps of measuring the amount of a CBL biomarker in a first sample of the cell, tissue, or organism; measuring the amount of a CBL biomarker in a second sample of the cell, tissue, or organism; and identifying a change in the amount of CBL in the cell, tissue, or organism based on the change in the amount of the CBL biomarker detected in the sample.

The CBL biomarker, sample, and CBL are as described above. In certain embodiments, the first sample is obtained prior to administration of a CBL inhibitor. In certain embodiments, the first sample is obtained at the first administration of a CBL inhibitor. In certain embodiments, the first sample is obtained prior to administration of a CBL inhibitor, and the second sample is obtained at the first administration of a CBL inhibitor. In certain embodiments, the first sample is obtained at the first administration of a CBL inhibitor, and the second sample is obtained after the first administration of a CBL inhibitor. In certain embodiments, the measuring steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times.

The CBL biomarker, sample, and CBL are as described above. The CBL inhibitor can be any CBL inhibitor deemed suitable by the person of skill. The treatment can be a treatment of any CBL disease or disorder described herein. In certain embodiments, the CBL inhibitor is a CBL inhibitor described herein. In certain embodiments, the CBL inhibitor is Compound 23, described herein. In certain embodiments, a subsequent dose of the CBL inhibitor is adjusted or determined based on the measured CBL level. If the CBL level is higher than desired, a higher subsequent dose of the CBL inhibitor can be selected. If the CBL level is lower than desired, a lower subsequent dose of the CBL inhibitor can be selected. In certain embodiments, the measuring steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times. In certain embodiments, the administering steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times. In certain embodiments, the measuring and administering steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times.

In another aspect, provided herein are methods of treating a patient in need thereof with a CBL inhibitor. In certain embodiments, the methods comprise administering to a patient a first dose of a CBL inhibitor; measuring the amount of a CBL biomarker in a first sample of the cell, tissue, or organism; measuring the amount of a CBL biomarker in a second sample of the cell, tissue, or organism; and identifying a change in the amount of CBL in the cell, tissue, or organism based on the change in the amount of the CBL biomarker detected in the sample. In certain embodiments, the CBL is Cbl-b. In certain embodiments, the CBL is c-Cbl. In certain embodiments, the CBL is Cbl-b and c-Cbl. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70. In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is two of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is three of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is four of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is five of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is six of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70.

The CBL biomarker, sample, CBL, CBL inhibitor, and CBL disease or disorder are as described above. In certain embodiments, a subsequent dose of the CBL inhibitor is adjusted or determined based on the measured CBL level. If the CBL level is higher than desired, a higher subsequent dose of the CBL inhibitor can be selected. If the CBL level is lower than desired, a lower subsequent dose of the CBL inhibitor can be selected. In certain embodiments, the measuring steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times. In certain embodiments, the administering steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times. In certain embodiments, the measuring and administering steps are repeated 1, 2, 3, 4, 5, 10, 15, 20, 25, or more times.

In certain embodiments the methods further comprise administering a second dose of the CBL inhibitor, wherein the dose is selected based on the amount of CBL identified. In certain embodiments of the methods, the second dose is increased if the amount of the CBL biomarker in a sample is below a predetermined range; wherein the second dose is maintained if the amount of the CBL biomarker in a sample is within a predetermined range; and/or wherein the second dose is decreased if the amount of the CBL biomarker in a sample is above a predetermined range. In certain embodiments of the methods, the second dose is decreased if the amount of the CBL biomarker in a sample is below a predetermined range; wherein the second dose is maintained if the amount of the CBL biomarker in a sample is within a predetermined range; and/or wherein the second dose is increased if the amount of the CBL biomarker in a sample is above a predetermined range. In certain embodiments of the methods, the second dose is increased, decreased, or maintained relative to the first dose.

In certain embodiments of the methods, an increase in the amount of the CBL biomarker indicates a decrease in the amount of CBL. In certain embodiments of the methods, a decrease in the amount of the CBL biomarker indicates a decrease in the amount of CBL.

In certain embodiments, the methods are conducted in vitro. In certain embodiments, the methods are conducted ex vivo.

The CBL biomarker can be detected by any technique deemed suitable. In certain embodiments, the CBL biomarker is detected by immunoassay, western blotting, ELISA, mass spectrometry, immunohistochemistry or flow cytometry. In certain embodiments, proximal biomarkers of CBL inhibition is detected by flow cytometry.

The disease or condition can be any disease or condition deemed suitable to the person of skill. In certain embodiments, the disease or condition is a cancer. In certain embodiments, the disease or condition is a solid tumor. In certain embodiments, the disease or condition is a hematological cancer.

In particular embodiments, the CBL biomarker is selected from phosphorylated interleukin-2-inducible kinase (pITK), phosphorylated hematopoietic lineage cell-specific protein (pHS1), phosphorylated phospholipase C2 (pPLCγ2), phosphorylated phospholipase C1 (pPLCγ1), phosphorylated spleen tyrosine kinase (pSYK), and phosphorylated Zeta chain associated protein kinase 70 (pZAP70). In certain embodiments, the biomarker is selected from pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, pZAP70, and combinations thereof. In certain embodiments, the biomarker is pITK. In certain embodiments, the biomarker is pHS1. In certain embodiments, the biomarker is pPLCγ2. In certain embodiments, the biomarker is pPLCγ1. In certain embodiments, the biomarker is pSYK. In certain embodiments, the biomarker is pZAP70. In certain embodiments, the biomarker is pHS1 and pPLCγ2. In certain embodiments, the biomarker is pPLCγ2 and pZAP70. In certain embodiments, the biomarker is pHS1 and pZAP70. In certain embodiments, the biomarker is pHS1, pPLCγ2, and pZAP70. In certain embodiments, the biomarker is two of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is three of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is four of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is five of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70. In certain embodiments, the biomarker is six of pITK, pHS1, pPLCγ2, pPLCγ1, pSYK, and pZAP70.

3. CBL Compounds

In the methods and compositions, the CBL compound can be any CBL compound deemed suitable to the person of skill. In certain embodiments, the CBL compound is any compound described in WO 2006/136502, WO 2007/010217, WO 2008/070305, WO 2019/148005, WO 2020/210508, WO 2020/236654, WO 2020/264398, or WO 2021/021761, WO 2021/243471, WO 2022/169997, WO 2022/169998, WO 2022/217276, WO 2022/221704, WO 2022/272248, or WO 2023/036330, the contents of which are hereby incorporated by reference in their entireties.

The CBL inhibitor can be administered or delivered by any composition or means deemed suitable to the practitioner of skill. In certain embodiments, the CBL inhibitor is a small molecule, administered in a pharmaceutical composition. In certain embodiments, the CBL inhibitor is delivered by an antibody, for instance as an antibody-drug conjugate. In certain embodiments, the CBL inhibitor is delivered in a delivery vehicle.

In certain embodiments, the CBL inhibitor compound is a compound of Formula (I):

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- or a tautomer thereof, or a pharmaceutically acceptable salt thereof,
- wherein:

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- is

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- Z¹is CH or N;
- Z²is CH or N;
- R¹is —CF₃or cyclopropyl;
- R²is —CF₃or cyclopropyl;
- R³is H, C₁-C₂alkyl, or C₁-C₂haloalkyl;
- R⁴is H, C₁-C₆alkyl, C₁-C₆haloalkyl, 4- to 8-membered heterocyclyl, or C₃-C₆cycloalkyl, wherein the heterocyclyl or cycloalkyl groups are optionally substituted by 1-5 R⁶groups;
- or R³and R⁴are taken together with the carbon atom to which they are attached to form C₃-C₅cycloalkyl or 4- to 6-membered heterocyclyl, each of which is optionally substituted by 1-5 R⁶groups;
- R⁵is H, C₁-C₆alkyl, C₁-C₆haloalkyl, or C₃-C₆cycloalkyl;
- each R⁶is independently C₁-C₆alkyl, halo, hydroxy, —O—(C₁-C₆alkyl), —CN, C₁-C₆alkyl-CN, C₁-C₆alkyl-OH, or C₁-C₆haloalkyl;
- or two R⁶groups attached to the same carbon atom are taken together with the carbon atom to which they are attached to form a spiro C₃-C₆cycloalkyl or spiro 4- to 6-membered heterocyclyl; X is H, C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkyl-OH, C₁-C₆alkyl-CN, C₃-C₆cycloalkyl optionally substituted by 1-5 R⁸groups, or

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- is 4- to 7-membered heterocyclyl or 5- to 8-membered heteroaryl, each of which heterocyclyl or heteroaryl optionally contains 1-2 additional heteroatoms selected from the group consisting of N, O, and S, and each of which heterocyclyl or heteroaryl is optionally substituted by 1-5 R⁸groups;
- each R⁷is independently H, C₁-C₆alkyl, C₁-C₆alkyl-OH, or C₁-C₆haloalkyl;
- or two R⁷groups are taken together with the carbon atom to which they are attached to form a C₃-C₅cycloalkyl or 3- to 5-membered heterocyclyl; and
- each R⁸is independently halo, C₁-C₆alkyl, C₁-C₆alkyl-CN, C₁-C₆alkyl-OH, C₁-C₆haloalkyl, —CN, oxo, or —O(C₁-C₆alkyl);
- or two R⁸groups are taken together with the carbon atom or atoms to which they are attached to form a spiro or fused C₃-C₅cycloalkyl or 3- to 5-membered heterocyclyl.

In certain embodiments,

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(i.e., the Ring A moiety), is

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In certain embodiments,

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(i.e., the Ring A moiety), is

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In certain embodiments, Z¹is CH. In other embodiments, Z¹is N. In certain embodiments, R¹is —CF₃. In other embodiments, R¹is cyclopropyl. In certain embodiments, the Ring A moiety is

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In certain embodiments, Z²is CH. In other embodiments, Z²is N. In certain embodiments, R²is —CF₃. In other embodiments, R²is cyclopropyl. In certain embodiments, the Ring A moiety is selected from the group consisting of:

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