Assays for measuring binding of small-molecule PD-l1 inhibitors to PD-l1

Abstract

The present disclosure relates to the discovery of ELISA-based assays that allow for the qualitative and quantitative evaluation of PD-L1 inhibition by a small molecule inhibitor. A biological sample obtained from a subject who has been administered the small-molecule agent is incubated ex vivo with a PD-L1 expression stimulator; PD-L1 expressing cells are isolated from the incubated biological sample; the isolated cells are lysed so as to yield free protein therefrom; and PD-L1 levels are quantitated in the free protein from the lysed isolated cells, thus assessing in vivo PD-L1 inhibition by the small-molecule agent.

Description

[0001] TITLE OF THE INVENTION

[0002] Assays For Measuring Binding Of Small-Molecule PD-L1 Inhibitors To PD-L1

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 532,166, filed August 11, 2023, which is incorporated herein by reference in its entirety.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Programmed death ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 plays a major role in suppressing the adaptive arm of immune system during pregnancy, tissue allograft transplants, autoimmune disease, and hepatitis. PD-L1 induces inhibitory signals through the interaction with programmed cell death- 1 (PD-1) expressed on the cell surface of T cells. This PD-1 / PD-L1 interaction results in the suppression of tumor-specific T cell responses, functioning as a tumor immune evasion mechanism.

[0007] In addition, there is evidence that viral infections can also upregulate immune checkpoints such as PD-1 / PD-L1 as a mechanism to evade host immunity. Thus, immunotherapy targeting immune checkpoints could be used to break viral antigenic immune tolerance, resulting in restoration of natural immunity and clearance of infected cells. One viral infection with the clear development of antigenic tolerance is chronic hepatitis B. In hepatitis B patients, hepatitis B virus-specific T and B cells are dampened, in part due to high antigenemia and upregulation of PD-1 (T and B cells) and PD-L1 (hepatocytes and antigen presenting cells (APCs)).

[0008] In another aspect, immune checkpoint inhibitors targeting the PD-1 / PD-L1 interaction have become a successful immunotherapy in treating many advanced cancers. The currently studied inhibitors of this interaction are monoclonal antibodies that bind to and directly disrupt the PD-1 / PD-L1 interaction. Indeed, three such aPD-Ll antibodies — atezolizumab, durvalumab, and avelumab — have been approved by the FDA for the treatment of bladder cancer, non-small cell lung carcinoma, and / or Merkel cell cancer.

[0009] To date, the discovery of small-molecule PD-1 / PD-L1 inhibitors has lagged primarily due to the large interacting surface area and tight binding involved in the PD-1 / PD-L1 interaction. It has been postulated that the discovery of a PD-1 / PD-L1 small-molecule inhibitor would require these molecules to inhibit through a completely different mechanism of action. Nonetheless, the discovery of a PD-L1 small-molecule checkpoint inhibitor could dramatically advance the field of checkpoint modulation and possibly lead to the development of therapeutics with better clinical properties than current antibody treatments.

[0010] Successful drug development requires demonstration of the investigational drug’s efficacy in vivo, such as in man. So far, checkpoint inhibitors investigated for in vivo activity have been antibodies. As such, it is not surprising that current methods to evaluate the degree of target engagement or target occupancy by a PD-L1 inhibitor, and / or confirm PD-L1 inhibitor activity in man, are based on the process of antibodies inhibiting PD-1 / PD-L1 interactions. Unfortunately, such methods are not suitable for the evaluation of target engagement / target occupancy mediated by small-molecule PD-L1 inhibitors. Such inhibitors would disrupt PD-1 / PD-L1 interactions by novel mechanisms and would not have the antibody structural elements required by the current methods to evaluate clinical PD-L1 inhibitor activity.

[0011] In addition, current methods for measuring PD-L1 inhibitor activity in man are far from convenient and very costly, as they require specialized equipment (in the form of flow cytometers) and specialized reagents (such as fluorescently labelled specific clones of antibodies to visualize and measure the target checkpoint protein on the flow cytometer). As such, new methods that are more sensitive and cost-effective for evaluating clinical activity of small-molecule PD-L1 inhibitors are needed.

[0012] Thus, there is a need in the art for compositions and methods that can be used to rapidly identify and characterize a small-molecule agent that binds to and inhibits the activity of PD-L1. In one aspect, novel sensitive and cost-effective for evaluating clinical activity of small-molecule PD-L1 inhibitors are needed. The present disclosure satisfies this unmet need.

[0013] BRIEF SUMMARY OF THE INVENTION

[0014] The present disclosure provides a method for assessing in vivo PD-L1 inhibition by a small-molecule agent in a subject. In certain embodiments, the method comprises incubating ex vivo a PD-L1 expression stimulator with a biological sample obtained from a subject who has been administered the small-molecule agent. In certain embodiments, the method comprises isolating PD-L1 expressing cells from the incubated biological sample. In certain embodiments, the method comprises lysing the isolated cells so as to yield free protein therefrom. In certain embodiments, the method comprises quantitating PD-L1 levels (if any) in the free protein from the lysed isolated cells. In certain embodiments, in vivo PD-L1 inhibition by the small-molecule agent is assessed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following detailed description of selected embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, selected embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0016] FIGs. 1 A-1C illustrate the finding that a non-limiting assay of the disclosure provides comparable small-molecule PD-L1 inhibition data as compared to a standard flow cytometrybased assay used in current clinical methods for measuring PD-L1 inhibitor activity. FIG. 1 A illustrates non-limiting level of PD-L1 measured on the monocyte population of peripheral blood mononuclear cells (PBMCs) after ex vivo treatment with a small-molecule PD-L1 inhibitor or dimethyl sulfoxide vehicle control conducted using a flow cytometry method. “Null” sample is of PBMCs without TruCulture tube stimulation and represents the limited sensitivity of the current methodology to detect effect of small-molecule inhibitor treatment. “T Stim” and “aCD3 / aCD28” samples are of PBMCs with TruCulture tube stimulation to enhance sensitivity of detecting small-molecule PD-L1 inhibitor effects. As shown in these figures, the histogram for monocytes expressing PD-L1 with small-molecule PD-L1 inhibitor treatment is shifted to the left when compared to DMSO vehicle control, indicating a decrease in PD-L1 cell surface expression. FIG. IB illustrates non-limiting calculated percentage of PD-L1 loss on the cell surface due to the activity of the small-molecule inhibitor treatment as described in FIG. 1 A. Treatment with the small-molecule PD-L1 inhibitor mediates internalization of PD-L1 expressed on the cell surface, thereby resulting in decreased PD-L1 cell surface expression. This is calculated by the equation: 100 (mean fluorescence intensity of small-molecule treated sample / mean fluorescence intensity of DMSO vehicle treated sample). FIG. 1C illustrates PD-L1 levels of “Null”, and TruCulture tube-stimulated samples “Tstim” and “aCD3 / aCD28” treated or not treated with small-molecule PD-L1 inhibitor as measured by ELISA. Upon treatment with small-molecule PD-L1 inhibitor, a decrease in the levels of PD-L1 as measured by ELISA is observed, and this effect is enhanced by stimulation with TruCulture tubes. These decreases in PD-L1 can be calculated as percentage of PD-L1 loss relative to the “Null”, “Tstim” or “aCD3 / CD28” conditions treated with DMSO vehicle as:

[0017] 100- (level ofPD-Ll in small-molecule treated sample / level ofPD-Ll in DMSO vehicle treated sample). FIGs. 2A-2B illustrate small-molecule PD-L1 inhibition data obtained with a nonlimiting assay of the disclosure and a standard flow cytometry-based assay. FIG. 2A illustrates the PD-L1 ELISA assay readout. FIG. 2B illustrates the PD-L1 flow cytometry assay readout.

[0018] FIG. 3 comprises a graph illustrating that a non-limiting assay of the disclosure provides a linear response to small-molecule PD-L1 inhibition and correlates with the response from a flow cytometry-based assay.

[0019] DETAILED DESCRIPTION

[0020] In one aspect, the present disclosure relates to the discovery of ex vivo ELISA-based assays that allow for the qualitative and / or quantitative evaluation of PD-L1 inhibition by any molecule, such as but not limited to a small-molecule inhibitor. In certain embodiments, the molecule is a small-molecule inhibitor.

[0021] In certain embodiments, the assays of the disclosure are based on a new method to evaluate the degree of PD-L1 inhibitor target engagement. In certain embodiments, the assays of the disclosure utilize distinct processes and methods for measuring PD-L1 levels on cells as compared to the flow cytometry methods used in the art. In certain embodiments, the assays of the disclosure are specific to the mechanism of small-molecule inhibitors of PD-L1, relying on the degradation of the PD-L1 protein to determine a target engagement effect. Such internalization of PD-L1 is not operative with antibody inhibitors of the PD-1 / PD-L1 interaction.

[0022] In certain embodiments, the assays of the disclosure are more sensitive compared to flow cytometry-based methods. In certain embodiments, the assays of the disclosure measure the levels of PD-L1 on all cells present in a blood sample, as compared to the flow cytometry methods used in the art which require identification of specific cell types. In certain embodiments, the assays of the disclosure incorporate an ex vivo incubation step that allows for enhancing PD-L1 expression and detection of compound effects.

[0023] In certain embodiments, the assays of the disclosure provide simpler processing steps than flow cytometry-based methods. The assays of the disclosure use a completely contained ex vivo cell system, which simplifies handling at clinical sites. Further, data analysis for the assays of the disclosure does not need to identify specific immune cell populations, unlike flow cytometry-based methods.

[0024] In certain embodiments, the assays of the disclosure have ease of readout. In certain embodiments, the assays of the disclosure do not require specialized equipment (unlike flow cytometers). In certain embodiments, the assays of the disclosure require a plate reader. In certain embodiments, the assays of the disclosure have the potential for greater reproducibility across clinical laboratories, in view of the simplified and streamlined protocol involved.

[0025] Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0027] As used herein, each of the following terms has the meaning associated with it in this section.

[0028] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0029] As used herein, the articles “a” and “an” refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0030] The term “assessing” includes any form of measurement, and includes determining if an element is present or not. The terms “determining,” “measuring,” “evaluating,” “assessing,” and “assaying” are used interchangeably and include quantitative and qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present, and / or determining whether it is present or absent.

[0031] “Detect” refers to identifying the presence, absence, level, or concentration of an agent.

[0032] As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.

[0033] As used herein, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.

[0034] As used herein, the term “Elisa” or “ELISA” refers to Enzyme-Linked Immunosorbent Assay, which is a sensitive labeled immunoassay that can be used to detect and / or quantify substances, including antibodies, antigens, proteins, glycoproteins, and / or hormones. Elisa uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured.

[0035] As an example, an antibody specific to an antigen is attached to a surface. Antigens from the sample to be tested are applied over the surface so that the antigens can bind to the antibody. Then, a different antibody specific to the antigen is applied over the surface so it can bind the antigen. This second antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme’s substrate is added. If binding between the antigen and the antibody takes place, the subsequent reaction produces a detectable signal, most commonly a color change.

[0036] An ELISA assay requires at least one antibody with specificity for a particular antigen. The sample comprising an antigen is immobilized on a solid support (usually a polystyrene microtiter plate, typically in a 96-well plate configuration) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed using a buffer, such as phosphate-buffered saline (PBS) and a non-ionic detergent, to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which quantitates the antigen in the sample.

[0037] In the ELISA protocol, usually, a serial dilution of concentrations is placed in the wells of the plate. After the results are measured, a standard curve from the serial dilutions data is plotted with a concentration on the x-axis using a log scale and absorbance on the y- axis using a linear scale.

[0038] There are thus four main general steps to completing an ELISA immunoassay: Coating (with either antigen or antibody), Blocking (typically with the addition of bovine serum albumin or BSA), Detection, and Final read. Detection is generically carried out using a substrate that can generate a color. The most commonly used substrates are horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The substrate for HRP is hydrogen peroxide and results in a blue color change. ALP measures the yellow color of nitrophenol and usually uses P-Nitrophenyl-phosphate (pNPP) as its substrate.

[0039] There are four major types of ELISA:

[0040] (a) Direct ELISA: The plate is initially coated with antigen and then washed to remove any potential unbound antigen. Any unbound sites on the ELISA plate are blocked using agents like BSA, ovalbumin, aprotinin, or other animal proteins. Then, a selected enzyme-conjugated primary detection antibody is added, binding directly to the antigen of interest. Next, the plate is rewashed to remove any unbound antibody and treated with a substrate / chromophore, such as alkaline phosphatase (AP) or Horseradish Peroxidase (HRP), which results in a color change. Direct ELISA eliminates secondary antibody cross-reactivity and has fewer steps than indirect ELISA.

[0041] (b) Indirect Elisa: The plate is initially coated with antigen and then washed to remove any potential unbound antigen. Any unbound sites on the ELISA plate are blocked using agents like BSA, ovalbumin, aprotinin, or other animal proteins. Then, a primary detection antibody that sticks to the antigen of interest is added, binding directly to the antigen. After washing, a secondary enzyme-linked antibody complementary to the primary antibody is added. Next, the plate is rewashed to remove any unbound antibody and treated with a substrate / chromophore, such as AP or HRP, which results in a color change. The indirect ELISA has higher sensitivity, lower cost, and higher flexibility than the direct ELISA.

[0042] (c) Sandwich ELISA: In this variation, the antigen is sandwiched between two layers of antibodies (capture and detection antibodies). For that, the wells of the plate are coated with a capture antibody, washed with buffer, and buffered / blocked with BSA. The antigen of interest is then added to the plates to bind to the capture antibody. The plate is rewashed, and the primary detection antibody is then added to the plate, followed by a buffer wash. Then the secondary enzyme-conjugated antibody is added. The plate is rewashed, and the substrate is added to produce a color change. The sandwich ELISA has the highest sensitivity among all the ELISA types.

[0043] (d) Competitive ELISA: This variation tests for the presence of an antibody specific for antigens in the test serum. This type of ELISA utilizes two specific antibodies: an enzyme-conjugated antibody and another antibody present in the test serum (if the serum is positive), which compete for binding to antigen. The presence of a color change means that the test is negative because the enzyme-conjugated antibody bound the antigens (not the antibodies of the test serum). The absence of color indicates a positive test and the presence of antibodies in the test serum.

[0044] As used herein, the term “Hepatitis B virus” (or HBV) refers to a virus species of the genus Orthohepadnavirus, which is a part of the Hepadnaviridae family of viruses, and that is capable of causing liver inflammation in humans.

[0045] As used herein, the term “Hepatitis D virus” (or HDV) refers to a virus species of the genus Deltaviridae, which is capable of causing liver inflammation in humans. The HDV particle comprises an envelope, which is provided by HBV and surrounds the RNA genome and the HDV antigen. The HDV genome is a single, negative stranded, circular RNA molecule nearly 1.7 kb in length. The genome contains several sense and antisense open reading frames (ORFs), only one of which is functional and conserved. The RNA genome is replicated through an RNA intermediate, the antigenome. The genomic RNA and its complement, the antigenome, can function as ribozymes to carry out self-cleavage and selfligation reactions. A third RNA present in the infected cell, also complementary to the genome, but 800 bp long and polyadenylated, is the mRNA for the synthesis of the delta antigen (HDAg).

[0046] As described herein, the term “PD-L1 inhibitor” includes any compound that is capable of inhibiting the expression and / or function of the protein Programmed Death-Ligand 1 (PD-L1) either directly or indirectly. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that plays a major role in suppressing the adaptive arm of immune system during pregnancy, tissue allograft transplants, autoimmune disease, and hepatitis. PD-L1 suppression of host immune responses to cancer or certain chronic viral infections has also been shown to play a role in promoting cancer or certain chronic viral infection growth or maintenance. PD-L1 binds to its receptor, the inhibitory checkpoint molecule PD-1 (which is found on activated T cells, B cells, and myeloid cells) so as to modulate activation or inhibition of the adaptive arm of immune system. In certain embodiments, the PD-L1 inhibitor inhibits the expression and / or function of PD-L1 by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

[0047] PD-L1 expressed by tumor cells plays a critical role in the induction of inhibitory signals through the interaction with programmed cell death-1 (PD-1), which is expressed on the surface of T cells. This PD-1 / PD-L1 interaction results in suppression of tumor-specific T cell responses functioning as a tumor immune evasion mechanism. Immune checkpoint inhibitors targeting the PD-1 / PD-L1 interaction have become a successful immunotherapy in treating many advanced cancers and are based on a mechanism of monoclonal antibody binding to and directly disrupting the PD-1 / PD-L1 interaction. Three of such antibodies have been approved by the FDA (z.e., atezolizumab, durvalumab, and avelumab).

[0048] Thus, PD-1 / PD-L1 binding has the net effect of inhibiting T cell receptor signaling, and the disruption and / or inhibition of this interaction represents a validated target for the treatment of many advanced cancers.

[0049] Reported PD-L1 inhibitors include, but are not limited to, compounds recited in one of the following patent application publications: US 2018 / 0057455; US 2018 / 0057486; WO 2017 / 106634; WO 2018 / 026971; WO 2018 / 045142; WO 2018 / 118848; WO 2018 / 119221; WO 2018 / 119236; WO 2018 / 119266; WO 2018 / 119286; WO 2018 / 121560; WO 2019 / 076343; WO 2019 / 087214; and are incorporated herein in their entirety by reference.

[0050] In certain embodiments, a cancer is amenable to treatment by inhibiting PD-1, PD-L1 or the PD-1 / PD-L1 interaction. In other embodiments, the cancer is at least one of pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. In yet other embodiments, the cancer is at least one of lymphoma, multiple myeloma, or leukemia. In yet other embodiments, the cancer is at least one of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), chronic myeloid leukemia (CML), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma, Waldestrom's macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL).

[0051] As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.

[0052] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, z.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0053] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and / or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.

[0054] As used herein, a “pharmaceutically effective amount,” “therapeutically effective amount,” or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0055] The term “prevent,” “preventing,” or “prevention” as used herein means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.

[0056] As used herein, a “small-molecule” inhibitor has a molecular weight that is equal to or lower that about 3,000 Daltons, 2,900 Daltons, 2,800 Daltons, 2,700 Daltons, 2,600 Daltons, 2,500 Daltons, 2,400 Daltons, 2,300 Daltons, 2,200 Daltons, 2,100 Daltons, 2,000 Daltons, 1,900 Daltons, 1,800 Daltons, 1,700 Daltons, 1,600 Daltons, 1,500 Daltons, 1,400 Daltons, 1,300 Daltons, 1,200 Daltons, 1,100 Daltons, 1,000 Daltons, 900 Daltons, 800 Daltons, 700 Daltons, 600 Daltons, 500 Daltons, 400 Daltons, 300 Daltons, and / or 200 Daltons.

[0057] By the term “specifically bind” or “specifically binds” as used herein is meant that a first molecule preferentially binds to a second molecule (e.g., a particular receptor or enzyme), but does not necessarily bind only to that second molecule.

[0058] As used herein, the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

[0059] The terms “treat,” “treating,” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

[0060] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partial numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0061] Assays In one aspect, the present disclosure relates to a method of evaluating PD-L1 inhibition by an inhibitor, such as but not limited to a small-molecule inhibitor. In certain embodiments, the inhibitor is a small-molecule inhibitor.

[0062] In certain embodiments, the method comprises contacting PD-L1 -expressing cells of the subject with the inhibitor (or a control compound) in vivo. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the PD-L1 expressing cells are contacted with the inhibitor (or a control compound) in vivo through treatment of the subject with the inhibitor (or control compound) using any route(s) of administration known in the art (oral, intravenous, subcutaneous, and so forth). In certain embodiments, PD-L1 inhibition by the small molecule inhibitor leads to internalization and degradation of the PD-L1 within the cell.

[0063] The PD-L1 expressing cells contacted with the inhibitor are then collected from the inhibitor-treated subject, so as to generate a biological sample. In certain embodiments, the biological sample comprises a blood and / or tissue sample from the subject.

[0064] The subject can be any subject known to a person of skill in the art. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human mammal. The biological sample can be obtained from the subject using any method known to a person of skill in the art.

[0065] In certain embodiments, the biological sample is obtained from a subject who has been administered the compound in vivo. In certain embodiments, after compound administration to the subject, the biological sample is withdrawn from the subject and then split into two samples. In certain embodiments, the first sample is used as-is or is incubated with an inert material, such as a vehicle, excipient, or carrier, such as DMSO. In certain embodiments, this first sample provides a measure of the biological effect of the compound that was administered to the subject in vivo. In certain embodiments, the second sample is incubated with a saturating amount of the small molecule inhibitor. In certain embodiments, such incubation induces maximal effect of PD-L1 inhibition and degradation. In certain embodiments, this second sample provides an internal reference to confirm quality of the biological sample and / or to account for any differences in the biological collection process. In certain embodiments, this second sample (i.e. maximal response) serves as a normalizer when evaluating biological samples drawn over a time course from the compound-treated subject (hence the equation under “Calculating Percentage of Target Occupancy / PD-L1 Loss Mediated by Small-Molecule Inhibitor Treatment”). In certain embodiments, the biological sample is contacted with a stimulator ex vivo. In certain embodiments, contacting the biological sample with the stimulator increases the amount / level of PD-L1 present in cells of the biological sample. In certain embodiments, a stimulator is an agent, such as for example small-molecule compounds, proteins, and / or antibodies, which promotes immune cell activation and / or proliferation when contacted with a biological sample. Non-limiting examples of stimulators include T cell stimulators, such as but not limited to anti-CD3 and / or anti-CD28 antibodies, and / or T cell activating antigens, such as but not limited to staphylococcal enterotoxin B or cytokines, such as but not limited to Interferon gamma. The duration, temperature, and buffer of incubation can be varied in order to modulate the immune cell activation and / or proliferation. In certain embodiments, the biological sample is incubated with the stimulator. In certain embodiments, the biological sample is incubated with the stimulator at physiological temperature (non-limiting example is 37°C). In certain embodiments, the biological sample is incubated with the stimulator at a temperature of about 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and / or 60 °C. In certain embodiments, the biological sample is incubated with the stimulator for at least about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, or longer. In certain embodiments, the incubation generates immune cell activation and proliferation that increases levels / amounts of PD-L1. In certain embodiments, measurable amounts of PD-L1 are considered to be any level that is above the lower limit of quantitation in the assay (greater than 25 pg / mL).

[0066] After incubation, the incubated biological sample can be further processed, for example by removing plasma from the sample and / or removing red blood cells from the sample. For example, cells within the incubated biological sample can be separated from plasma by insertion of a valve separator into the tubes, which forces cells to the bottom of the tubes and leaves plasma on the other side of the valve. Plasma can then be decanted or removed by pipetting, after which the valve separator is removed. The cells can be subjected to red blood cell removal, for example by incubation with a red blood cell lysis buffer that selectively removes red blood cells (for example, ThermoScientific Catalog No. 00-4300-54 or equivalent). In non-limiting embodiments, such buffers typically contain ammonium chloride, which preferentially lyses non-nucleated cells such as red blood cells. After incubation with a red blood cell lysis buffer, remaining cells can be washed with phosphate buffered saline and cells are centrifuged to yield a cell pellet. The incubated sample, after plasma removal and / or red blood cell lysis, can then be submitted to a cell lysis process, using any methods known in the art. For example, cell pellets isolated after red blood cell lysis and washes can be resuspended in a volume of cell lysis buffer (example: Sigma-Aldrich Catalog No. 4719964001). Samples can be incubated and centrifuged / decanted to yield clarified supernatants. Supernatants can then be transferred to appropriately sized tubes for protein content analysis and PD-L1 measurement.

[0067] The amount of protein derived from lysed biological sample can be measured in a protein measurement assay, using any methods and / or kits known in the art. One example of such an assay is the bicinchoninic acid (BCA) assay, which combines the reduction of Cu2+to Cu1+by protein in an alkaline medium. The assay further comprises colorimetric detection of the cuprous cation Cu1+by bicinchoninic acid. Reaction of the bicinchoninic acid with the reduced Cu1+cation results in formation of a purple-colored reaction product that exhibits a linear absorbance at 562 nm with increasing protein concentrations. Comparison of the tested sample’s absorbance values at 562 nm with a standard curve generated by measurement of a protein with known concentration such as bovine serum albumin allows quantitation of protein content levels in the tested sample.

[0068] Amounts of protein from each tested sample are then analyzed for PD-L1 levels using any methods and / or kits known in the art. One example of such an assay to measure PD-L1 levels is R&D Systems Catalog No.DB7H10. Samples to be assessed may be inputted into this assay either without dilution or diluted with phosphate-buffered saline to achieve the targeted protein levels. Samples are quantified by interpolation from a standard curve of recombinant PD-L1 protein.

[0069] In a non-limiting example, a commercially available TruCulture tube system can be used for the ex vivo incubation of the assays of the disclosure. For example, blood can be drawn into TruCulture tubes spiked with the compound under testing as a positive control reference used as a normalizer to account for variability in the biological sample collection when multiple samples are drawn over a time course, or spiked with a buffer and / or DMSO as the test sample (in this case the amount of small-molecule inhibitor present in the sample is derived from the prior in vivo administration of the drug to the subject), or an internal reference (for example, a known small-molecule PD-L1 inhibitor). The blood sample is mixed and incubated, then plasma is separated and removed from the incubated blood sample. The resulting cells undergo red blood cell removal, and then are lysed for total protein. PD-L1 levels in the sample can be determined using any known protein quantitation method, such as commercial kits (for example, Catalogue # DB7H10, R&D Systems). EXPERIMENTAL EXAMPLES

[0070] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0071] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out illustrative embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0072] Example 1:

[0073] Preparation of TruCulture Tubes (performed in a sterile biosafety cabinet):

[0074] 1. Set block heater to 37°C.

[0075] 2. Thaw the required number of TruCulture tubes (2 per timepoint). The tubes can also be thawed overnight at 2-8°C. In certain non-limiting embodiments, the culture medium within the tube comprises one or more stimulators. In certain non-limiting embodiments, the culture medium within the tube does not comprise one or more stimulators, and one or more stimulators are contacted with the biological sample at any time between biological sample collection (step 12) and incubation (step 16).

[0076] 3. To spike the first TruCulture tube, disinfect the rubber septum of a vial containing a 0.1% DMSO in phosphate-buffered saline solution (test sample) with an alcohol pad and use a sterile 1 mL with 25-28g needle and draw up >0.2 mL of reagent from the vial.

[0077] 4. Disinfect the rubber septum in the screw cap end (top-end) of the first TruCulture tube with an alcohol pad provided in kit. Do not use disinfectants based on iodine or formaldehyde. Pierce the rubber septum with the syringe and as accurately as possible, inject 0.2 mL of solution into the first TruCulture tube. Remove the syringe and mix the tube contents by inverting 3 times, end over end. Avoid foaming.

[0078] 5. To spike the second TruCulture tube, disinfect the rubber septum of vial containing 16 pM of small-molecule inhibitor (positive control reference) with an alcohol pad and use a sterile 1 mL syringe graduated with 25-28g needle and draw up >0.2 mL of reagent from the vial.

[0079] 6. Disinfect the rubber septum in the screw cap end (top-end) of the second TruCulture tube with an alcohol pad provided in kit. Do not use disinfectants based on iodine or formaldehyde.

[0080] 7. Pierce the rubber septum with the syringe and as accurately as possible, inject 0.2 mL of solution into the second TruCulture tube. Remove the syringe, mix the tube contents by inverting 3 times, end over end. Avoid foaming. This will yield a final concentration of 1 pM of small-molecule inhibitor in the TruCulture tube. The final concentration of the small-molecule inhibitor can be adjusted such that the concentration of the small-molecule inhibitor to be used results in a maximal effect of PD-L1 loss in the sample.

[0081] 8. Prepared TruCulture tubes are used to collect blood from subjects administered the small-molecule inhibitor drug by any method known in the art.

[0082] Blood Sample Collection:

[0083] 9. Pull the plunger down in each prepared TruCulture tube until the plunger “clicks” or licks in place to create a vacuum and make space for the blood.

[0084] 10. Use a butterfly needle for venipuncture or an indwelling intravenous catheter. An adaptor such as Sarstedt, catalog no. 14.1205 is attached and used to connect the butterfly needle or indwelling intravenous catheter to the prepared TruCulture tubes.

[0085] 11. Use an empty tube to prime the butterfly needle tubing (fill tubing with blood) or clean the intravenous line of the indwelling intravenous catheter.

[0086] 12. Remove the empty tube and replace with a prepared TruCulture tube from step 4. The TruCulture tube should automatically fill due to the vacuum created in step 9. Allow tube to completely fill, about 1 mL of blood directly into the TruCulture tube for a total of 3 mL. Invert tube gently 3-5 times end-over-end and avoid foaming.

[0087] 13. Repeat step 12 with the second TruCulture tube from step 7.

[0088] 14. Break away syringe plunger on each TruCulture tube.

[0089] 15. Gently tap the TruCulture tubes on a hard surface to remove liquid from the screw cap before placing on the heat block. Place tubes (screw cap end up) in a 37°C-heat block within 15 minutes of adding the blood. Record time placed on heat block.

[0090] 16. Incubate overnight (18-20 hours). Remove tubes from heat block and record time removed from heat block. 17. In a clean, sterile tissue culture hood, assemble plunger / valve separator immediately before insertion into TruCulture tube.

[0091] 18. TruCulture tube should remain upright to avoid disturbing cells. Screw the cap off TruCulture tube and insert an assembled plunger / valve separator to separate the cells from the supernatant / plasma. Leave ~2 mm space between the valve separator and cells.

[0092] 19. Remove plunger by twisting counter-clockwise. Leave the Seraplas valve filter in the sample and pull out stick. Discard stick.

[0093] 20. Re-cap TruCulture tube with screw cap.

[0094] 21. Store TruCulture tubes at 2-8°C until shipping to laboratory for sample processing or proceed with sample processing.

[0095] Sample Processing:

[0096] 22. Prepare IX Red Blood Cell (RBC) Lysis Buffer.

[0097] 23. For each cell pellet being processed, prepare 1 Lysis Tube by adding 20.0 mL of IX RBC Lysis Buffer to a 50 mL conical tube, 1 Large Wash Tube by adding 10.0 mL of Dulbecco’s phosphate-buffered saline (DPBS) to a 15 mL conical tube, and 1 Centrifugation Tube (which is a 1.5 mL or 2 mL polypropylene tube that remains empty until further instructed), labelling each tube appropriately.

[0098] 24. Add Cell Pellet to the associated Lysis Tube by performing the following: Unscrew the cap of the TruCulture tube and discard the supernatant. Then insert a rod, rotate it in place to the valve, pull the valve out and discard. Resuspend the pellet in the TruCulture tube by pipetting as much volume as feasible up and down 3 - 4 times and transfer 1.00 mL of the Cell Pellet to the associated Lysis Tube and rinse the pipette tip by pipetting as much volume as feasible up and down 5 times. Upon addition, gently invert each Lysis Tube 3 - 5 times to mix.

[0099] 25. Place Lysis Tube(s) on the bench-top and incubate at RT for 10 - 30 minutes then place in a centrifuge.

[0100] 26. Centrifuge for 5 minutes at 450 x g at RT.

[0101] 27. Remove Lysis Tube(s) from centrifuge. Carefully remove supernatant by pouring through a funnel into biohazard waste container containing disinfectant or via pipetting. If pouring, then with the Lysis Tube still inverted, lightly shake it mid-air 2 - 3 times to remove as much supernatant as you can without disturbing the pellet.

[0102] 28. Resuspend each pellet with 1.00 mL of DPBS using a pipette and transfer cells to the Wash Tube already containing 10 mL of DPBS. Rinse the pipette tip by pipetting as much volume as feasible up and down 5 times. Gently invert each wash tube 3 - 5 times to mix.

[0103] 29. Centrifuge for 5 minutes at 450 x g at room temperature.

[0104] 30. Remove Wash Tube(s) from centrifuge. Carefully remove supernatant by pouring it into biohazard waste container containing disinfectant using a funnel or via pipetting. If pouring, then with the Wash Tube still inverted, lightly shake it mid-air 2 - 3 times to remove as much supernatant as possible without disturbing the pellet.

[0105] 31. Repeat Steps 28 and 29, except by adding 10 mL of DPBS directly to the Wash Tube rather than transferring to a new tube.

[0106] 32. Upon completion of centrifugation, remove the Wash Tube(s) from centrifuge and, without disturbing the cell pellet, discard supernatant by slowly pipetting it from the top leaving a thin but visible layer of DPBS in the tube. Using an appropriately sized pipette, carefully remove as much of the remaining DPBS from cell pellet as is feasible.

[0107] 33. Resuspend cell pellet with 300 pL of Cell Lysis Buffer (example: Sigma-Aldrich, catalog no. 4719964001). Incubate on a plate shaker (-450 rpm) at RT for 10 - 20 minutes. Using an appropriately sized pipette, transfer the entirety of the resulting cell lysate to a Centrifugation Tube.

[0108] 34. Centrifuge for 15 minutes at 14,000 x g at 4 ± 2°C.

[0109] 35. Remove from the centrifuge and, taking from the supernatant, prepare one aliquot of 290 pL in screw-cap cryotube, then store Human WBC Lysates at < -80°C pending protein content and PD-L1 level analysis.

[0110] Protein Content Analysis:

[0111] 36. Prepare Standards, Controls and Blank samples per manufacturer’s protocol for the protein content assay kit (example: Pierce, catalog no. 23225, BCA kit).

[0112] 37. Pipette 25.0 pL / well in a 96-well microplate of each Standard, Control, Blank and undiluted sample from step 35.

[0113] 38. Pipette 200 pL / well of BCA Working Reagent into each assay well. Cover plate and briefly place on a plate shaker at -450 rpm to mix well.

[0114] 39. Transfer covered plate to incubator and incubate for 30 ± 2 min at 37 ± 2°C.

[0115] 40. After incubation, remove the plate and let sit at room temperature for a minimum of five minutes.

[0116] 41. Measure the absorbance at 562 nm on a plate reader within 30 minutes of removing from the incubator. 42. Interpolate sample protein concentrations based on standard curve.

[0117] 43. Equal amounts of protein ranging from 6-10 micrograms from each tested sample are then analyzed for PD-L1 levels.

[0118] PD-L1 Quantitation:

[0119] 44. Prepare Standards, Controls and Blank samples per manufacturer’s protocol for PD- L1 assay kit (example: R&D Systems, catalog no. DB7H10).

[0120] 45. Dilute samples to be assayed to be 0.06 to 0.1 pg / mL with DPBS.

[0121] 46. Add 50.0 pL / well of Assay Diluent RD1-41, and then 100 pL / well of Standards, Controls, blank, and samples. Seal plate and incubate at room temperature for 2 hr ±15min with shaking (-450 rpm). Protect from light.

[0122] 47. Wash plate 3X with 300 pL / well of Wash Buffer. Blot to remove excess buffer. Add 200 pL / well of Antibody Conjugate. Seal plate and incubate at RT for 2 hr±15min with shaking (-450 rpm). Protect from light.

[0123] 48. Prepare Substrate Solution. Wash plate 3X with 300 pL / well of Wash Buffer. Blot to remove excess buffer. Add 200 pL / well of Substrate Solution. Seal plate and incubate at RT for 30 ± 3 min with shaking (-450 rpm). Protect from light.

[0124] 49. Add 50.0 pL / well of Stop Solution and read absorbance on plate reader at 450 nm with a 570 nm reference.

[0125] 50. Interpolate sample PD-L1 levels based on standard curve.

[0126] Calculating Percentage of Target Occupancy / PD-Ll Loss Mediated by Small-Molecule Inhibitor Treatment:

[0127] The percentage of target occupancy or PD-L1 loss mediated by administration of a small-molecule inhibitor drug to a subject is calculated as follows:

[0128] PD-L1 levels in maximum e ffect positive control reference TruCulture tube x 100%

[0129] PD-L1 levels in buffer or DMSO test sample TruCulture tube

[0130] In certain non-limiting embodiments, the PD-L1 levels in the test sample (i.e. from the subject who has been administered the compound in vivo) are greater than the PD-L1 levels in the maximum effect positive control reference, at least because the compound treatment results in degradation of PD-L1. In certain non-limiting embodiments, as the PD- L1 levels in the test sample increase towards maximal effect, the % target occupancy will also increase, ultimately resulting in 100% target occupancy if the test sample PD-L1 levels are equal to the PD-L1 levels in the maximum effect positive control reference tube.

[0131] Enumerated Embodiments

[0132] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0133] Embodiment 1 provides a method for assessing in vivo PD-L1 inhibition by a smallmolecule agent in a subject. In certain embodiments, the method comprises incubating ex vivo a PD-L1 expression stimulator with a biological sample obtained from a subject who has been administered the small-molecule agent. In certain embodiments, the method comprises isolating PD-L1 expressing cells from the incubated biological sample. In certain embodiments, the method comprises lysing the isolated cells so as to yield free protein therefrom. In certain embodiments, the method comprises quantitating PD-L1 levels (if any) in the free protein from the lysed isolated cells. In certain embodiments, in vivo PD-L1 inhibition by the small-molecule agent is assessed.

[0134] Embodiment 2 provides the method of Embodiment 1, wherein the incubating step increases levels of PD-L1 expressed ex vivo in the PD-L1 expressing cells.

[0135] Embodiment 3 provides the method of any one of Embodiments 1-2, wherein the PD- L1 expression stimulator comprises a T cell stimulator and / or a T cell activating antigen.

[0136] Embodiment 4 provides the method of Embodiment 3, wherein the T cell stimulator comprises an anti-CD3 and / or anti-CD28 antibody.

[0137] Embodiment 5 provides the method of Embodiment 3, wherein the T cell activating antigen comprises staphylococcal enterotoxin B and / or a cytokine, optionally Interferon gamma.

[0138] Embodiment 6 provides the method of any one of Embodiments 1-5, wherein the biological sample comprises blood or tissue from the subject.

[0139] Embodiment 7 provides the method of any one of Embodiments 1-6, wherein the isolating step comprises centrifugation, filtration, and / or valve-assisted separation of cells from plasma.

[0140] Embodiment 8 provides the method of Embodiment 7, wherein the sample comprises blood, and wherein the isolating step comprises removing at least a fraction of the plasma of the sample. Embodiment 9 provides the method of any one of Embodiments 1-8, wherein the sample comprises blood, and wherein the isolating step further comprises removing at least a fraction of the red blood cells of the sample.

[0141] Embodiment 10 provides the method of any one of Embodiments 1-9, wherein the lysing step comprises treatment with a buffer containing a detergent and / or a protease inhibitor.

[0142] Embodiment 11 provides the method of any one of Embodiments 1-10, wherein the quantitating step is performed using an ELISA assay or any other immunoassay.

[0143] Embodiment 12 provides the method of any one of Embodiments 1-11, wherein the quantitating comprises comparing the PD-L1 levels measured when the subject is administered the small-molecule agent with the PD-L1 levels measured when an equivalent subject is administered a control.

[0144] Embodiment 13 provides the method of Embodiment 12, wherein the control is a positive control that inhibits PD-L1.

[0145] Embodiment 14 provides the method of Embodiment 13, wherein if the PD-L1 levels measured using the positive control are similar to the PD-L1 levels measured using the biological samples derived from a subject administered the small-molecule agent, then the small-molecule agent is determined to be an in vivo PD-L1 inhibitor.

[0146] Embodiment 15 provides the method of Embodiment 12, wherein the control is a known PD-L1 inhibitor, preferably a known small-molecule PD-L1 inhibitor.

[0147] Embodiment 16 provides the method of Embodiment 15, wherein if the PD-L1 levels measured using the known PD-L1 inhibitor at a given concentration are higher than the PD- L1 levels measured using the small-molecule agent at the same given concentration, then the small-molecule agent is determined to be a more potent PD-L1 inhibitor than the known PD- L1 inhibitor.

[0148] Embodiment 17 provides the method of Embodiment 15, wherein if the PD-L1 levels measured using the known PD-L1 inhibitor at a given concentration are lower than the PD-L1 levels measured using the small-molecule agent at the same given concentration, then the small-molecule agent is determined to be a less potent PD-L1 inhibitor than the known PD- L1 inhibitor.

[0149] Embodiment 18 provides the method of Embodiment 1, wherein the ex vivo incubation step uses a first incubation system, wherein the biological sample is used as-is and / or is incubated with an inert (non-PD-Ll inhibiting) material; and wherein the ex vivo incubation step uses a second incubation system, wherein the biological sample is incubated with a known small-molecule inhibitor in an amount sufficient to induce complete PD-L1 inhibition and degradation in the second incubation system.

[0150] Embodiment 19 provides the method of Embodiment 18, wherein the known smallmolecule inhibitor is the small-molecule inhibitor administered to the subject.

[0151] Embodiment 20 provides the method of the Embodiment 18, wherein the quantitation step comprises quantitating PD-L1 levels in the first incubation system to provide a first reading and wherein the quantitation step comprises quantitating PD-L1 levels in the second incubation system to provide a second reading.

[0152] Embodiment 21 provides the method of Embodiment 20, wherein the % target occupancy for the biological sample with respect to the small-molecule inhibitor is provided by the ratio: [second reading / first reading] x 100%.

[0153] Other Embodiments

[0154] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0155] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method for assessing in vivo PD-L1 inhibition by a small-molecule agent in a subject, the method comprising: incubating ex vivo a PD-L1 expression stimulator with a biological sample obtained from a subject who has been administered the small-molecule agent; isolating PD-L1 expressing cells from the incubated biological sample; lysing the isolated cells so as to yield free protein therefrom; and quantitating PD-L1 levels (if any) in the free protein from the lysed isolated cells; thus assessing in vivo PD-L1 inhibition by the small-molecule agent.

2. The method of claim 1, wherein the incubating step increases levels of PD-L1 expressed ex vivo in the PD-L1 expressing cells.

3. The method of claim 1, wherein the PD-L1 expression stimulator comprises a T cell stimulator and / or a T cell activating antigen.

4. The method of claim 3, wherein the T cell stimulator comprises an anti-CD3 and / or anti-CD28 antibody.

5. The method of claim 3, wherein the T cell activating antigen comprises staphylococcal enterotoxin B and / or a cytokine, optionally Interferon gamma.

6. The method of claim 1, wherein the biological sample comprises blood or tissue from the subject.

7. The method of claim 1, wherein the isolating step comprises centrifugation, filtration, and / or valve-assisted separation of cells from plasma.

8. The method of claim 7, wherein the sample comprises blood, and wherein the isolating step comprises removing at least a fraction of the plasma of the sample.

9. The method of claim 1, wherein the sample comprises blood, and wherein the isolating step further comprises removing at least a fraction of the red blood cells of the sample.

10. The method of claim 1, wherein the lysing step comprises treatment with a buffer containing a detergent and / or a protease inhibitor.

11. The method of claim 1, wherein the quantitating step is performed using an ELISA assay or any other immunoassay.

12. The method of claim 1, wherein the quantitating comprises comparing the PD-L1 levels measured when the subject is administered the small-molecule agent with the PD-L1 levels measured when an equivalent subject is administered a control.

13. The method of claim 12, wherein the control is a positive control that inhibits PD-L1.

14. The method of claim 13, wherein if the PD-L1 levels measured using the positive control are similar to the PD-L1 levels measured using the biological samples derived from a subject administered the small-molecule agent, then the small-molecule agent is determined to be an in vivo PD-L1 inhibitor.

15. The method of claim 12, wherein the control is a known PD-L1 inhibitor, preferably a known small-molecule PD-L1 inhibitor.

16. The method of claim 15, wherein if the PD-L1 levels measured using the known PD- L1 inhibitor at a given concentration are higher than the PD-L1 levels measured using the small-molecule agent at the same given concentration, then the small-molecule agent is determined to be a more potent PD-L1 inhibitor than the known PD-L1 inhibitor.

17. The method of claim 15, wherein if the PD-L1 levels measured using the known PD- L1 inhibitor at a given concentration are lower than the PD-L1 levels measured using the small-molecule agent at the same given concentration, then the small-molecule agent is determined to be a less potent PD-L1 inhibitor than the known PD-L1 inhibitor.

18. The method of claim 1, wherein the ex vivo incubation step uses a first incubation system, wherein the biological sample is used as-is and / or is incubated with an inert (non-PD-Ll inhibiting) material; and wherein the ex vivo incubation step uses a second incubation system, wherein the biological sample is incubated with a known small-molecule inhibitor in an amount sufficient to induce complete PD-L1 inhibition and degradation in the second incubation system.

19. The method of claim 18, wherein the known small-molecule inhibitor is the smallmolecule inhibitor administered to the subject.

20. The method of claim 18, wherein the quantitation step comprises quantitating PD-L1 levels in the first incubation system to provide a first reading and wherein the quantitation step comprises quantitating PD-L1 levels in the second incubation system to provide a second reading.

21. The method of claim 20, wherein the % target occupancy for the biological sample with respect to the small-molecule inhibitor is provided by the ratio:[second reading / first reading] x 100%.

22. The method of claim 1, wherein the subject is a mammal.

23. The method of claim 1, wherein the subject is a human.

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