WO2021247188A1

WO2021247188A1 - Cd73 antagonist potency assay and methods of use thereof

Info

Publication number: WO2021247188A1
Application number: PCT/US2021/031029
Authority: WO
Inventors: Hai Zhuan ZHANG; Diane L. VY; Darren KAMIKURA; John M. LEHRACH; Judit Wahlman JOHNSON; Lisa LUNDBERG; Marcel S. ZOCHER
Original assignee: Bristol-Myers Squibb Company
Priority date: 2020-06-05
Filing date: 2021-05-06
Publication date: 2021-12-09

Abstract

The present disclosure relates to methods of characterizing a CD73 antagonist, which method is suitable as a potency assay. The assay provided herein determines the ability of a CD73 antagonist to inhibit the CD73 -mediated enzymatic conversion of AMP to adenosine. In vitro assays, including those with a CD73 antagonist reference sample, are also provided.

Description

CD73 ANTAGONIST POTENCY ASSAY AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/035,342, filed June 5, 2020, the contents of which are hereby incorporated by reference.

BACKGROUND

Cluster of Differentiation 73 (CD73), also known as ecto-5'-nucleotidase (ecto-5'NT, EC 3.1.3.5), is a glycosyl-phosphatidylinositol (GPI)-linked cell surface enzyme found in most tissues, but particularly expressed in endothelial cells and subsets of hematopoietic cells (Resta et al., Immunol Rev 1998;161:95-109 and Colgan et al., Prinergic Signal 2006;2:351-60). CD73 is known to catalyze the dephosphorylation of extracellular nucleoside monophosphates into nucleosides, such as adenosine. Adenosine is a widely studied signaling molecule which mediates its biological effects through several receptors, including Al, A2A, A2B, and A3. Adenosine has been shown to regulate proliferation and migration of many cancers and to have an immunosuppressive effect through the regulation of anti-tumor T cells (Zhang et al., Cancer Res 2010;70:6407-11).

CD73 has been reported to be expressed on many different cancers, including colon, lung, pancreas, ovary, bladder, leukemia, glioma, glioblastoma, melanoma, thyroid, esophageal, prostate and breast cancers (Jin et al., Cancer Res 2010;70:2245-55 and Stagg et al., PNAS 2010;107:1547-52). Moreover, CD73 expression in cancer has been linked to increased proliferation, migration, neovascularization, invasiveness, metastesis and shorter patient survival. CD73 activity has also been proposed as a prognostic marker in papillary thyroid carcinomas. While CD73 has been shown to regulate cell-cell and cell-matrix interactions on tumor cells, CD73 expression and activity has also been linked to reduced T-cell responses and implicated in drug resistance (Spychala et al., Pharmacol Ther 3000;87: 161-73). Thus CD73 can regulate cancer progression both directly and indirectly, which highlights its potential as a therapeutic target.

Accordingly, methods for regulating CD73 activity are in development, such as the use of CD73 antagonists to inhibit or block CD73 activity. Therefore, methods for measuring the activity and potency of such antagonists are needed. For example, it is critical that different lots of the same CD73 antagonist have the same potency to ensure appropriate dosing in subjects. Accordingly, methods to accurately quantify the potency of a CD73 antagonist, e.g., prior to the use of the antagonist as a therapeutic agent, are highly desirable.

SUMMARY

The present disclosure provides methods for assessing, determining and/or quantifying the potency of a CD73 antagonist.

In some aspects, the disclosure provides a method for assessing the potency of a CD73 antagonist, comprising the steps of:

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist; and

(b) measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist.

In other aspects, the disclosure provides a method for quantifying the ability of a CD73 antagonist to prevent enzymatic conversion of AMP to adenosine by human CD73 expressed on a cell, comprising the steps of:

(b) measuring the amount of AMP after the period of time as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73.

In yet other aspects, the disclosure provides an in vitro assay for assessing the activity of a CD73 antagonist comprising the steps of:

(b) measuring the amount of AMP after the period of time as an indication of the activity of the CD73 antagonist. In some aspects, the disclosure provides a method for determining whether a manufactured CD73 antagonist fulfills a predefined criterion, e.g., having a similar potency to that of a reference standard CD73 antagonist, comprising the steps of:

(b) measuring the amount of AMP after the period of time.

In any of the foregoing or related aspects, the methods comprise permeabilizing the cells prior to measuring the amount of AMP. In some aspects, permeabilizing the cells comprises contacting the cells with a cell permeabiliztion agent, such as a detergent. In some aspects, the detergent is Triton™ X-100. In some aspects, the detergent is 0.5% to 5% Triton™ X-100. In some aspects, the detergent is 0.5% Triton™ X-100. In some aspects, Triton™ X-100 is 95%- 100% pure. In any of the foregoing or related aspects, the step of permeabilizing the cells further comprises shaking the cells after the addition of the cell permeabilization agent.

In any of the foregoing or related aspects, the step of measuring the amount of AMP is determined by (i) adding a first reagent comprising a first enzyme capable of converting the AMP to adenosine diphosphate (ADP); (ii) adding a second reagent comprising a second enzyme capable of converting the ADP to adenosine triphosphate (ATP); and (iii) determining the amount of ATP. In some aspects, the first and second reagents are added simultaneously or consecutively. In some aspects, the second reagent is added after AMP is converted to ADP.

In any of the foregoing or related aspects, the first enzyme is polyphosphate: AMP phosphotransferase (PAP) and the second enzyme is adenylate kinase (AK). In some aspects, the first and second reagents are Reagent I and AMP Detection Solution, respectively, from an Amp- Glo™ assay. In some aspects, the ratio of first reagent: second reagent is 1:2.

In any of the foregoing or related aspects, the step of determining the amount of ATP comprises adding luciferase and luciferin.

In any of the foregoing or related aspects, the cells are human cells. In some aspects, the cells naturally express human CD73 on the cell surface or the cells have been engineered to express human CD73 on the cell surface. In some aspects, the cells are SNU-387 cells (ATCC Accession No. CRL-2237) or Calu-6 cells (ATCC Accession No. HTB-56). In some aspects, about 10,000 cells expressing human CD73 are used per well of a 96 well plate. In some aspects, the cells are passaged no more than 30 times.

In any of the foregoing or related aspects, AMP has a concentration of about 10 μΜ to about 100 μΜ. In some aspects, AMP has a concentration of about 15 μΜ to about 35 μΜ. In some aspects, AMP has a concentration of about 25μΜ.

In any of the foregoing or related aspects, the method is performed at a temperature of

20°-27°C.

In any of the foregoing or related aspects, the method comprises generating a dose- response curve with the CD73 antagonist and a dose-response curve with a reference standard CD73 antagonist having a known potency, and determining the potency of the CD73 antagonist by comparing the dose- response curves. In some aspects, the reference standard CD73 antagonist is an anti-CD73 antagonist antibody which binds to human CD73. In some aspects, the reference standard CD73 antagonist is Ab A.

In some aspects, the disclosure provides a method for assessing the potency of a CD73 antagonist (or “test CD73 antagonist”), comprising the steps of:

(a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) measuring the amount of AMP after the period of time by:

(i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

(c) comparing the amount of AMP measured between the reference standard CD73 antagonist and the CD73 antagonist; wherein the amount of AMP determines the potency of the CD73 antagonist.

In other aspects, the disclosure provides a method for assessing the potency of a CD73 antagonist, comprising the steps of: (a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

(b) measuring the amount of AMP after (a) by:

(i) permeabilizing the cells with 0.5% Triton X-100 for 5 minutes while shaking;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I for about 60 minutes at

20°-27°C;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution for about 60 minutes at 20°-27°C; and

(iv) exposing the cells of (iii) to a luminescent measuring device;

(c) comparing the amount of AMP between the reference standard CD73 antagonist and the CD73 antagonist, wherein the amount of AMP determines the potency of the CD73 antagonist.

In yet other aspects, the disclosure provides a method for assessing the potency of a CD73 antagonist (or “test CD73 antagonist”), comprising the steps of:

(a) contacting 4, 5, 6, 7, 8 or more increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time, wherein the level of AMP is determined by:

(i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

(c) comparing the two dose-response curves to thereby assess the potency of the CD73 antagonist.

In further aspects, the disclosure provides a method for assessing the potency of a CD73 antagonist, comprising the steps of: (a) contacting 4, 5, 6, 7, 8 or more increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference CD73 antagonist, after the period of time, wherein the level of AMP is determined by:

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

In any of the foregoing aspects, step (a) comprises contacting 8 concentrations ranging from 2 μg/ml to 0.0033μg/mL of the CD73 antagonist and the reference standard CD73 antagonist with the cells expressing human CD73 and AMP.

In other aspects, the disclosure provides a kit for determining the potency of a CD73 antagonist, comprising cells expressing human CD73, AMP and instructions for contacting the CD73 antagonist with the cells and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist and subsequently measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist. In some aspects, the kit comprises a reference standard CD73 antagonist and instructions for preparing dose-response curves with the CD73 antagonist and the reference standard CD73 antagonist. In some aspects, reference standard antagonist is an anti-CD73 antagonist antibody. In some aspects, the anti-CD73 antagonist antibody is Ab A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing an overview of the CD73 antagonist potency assay disclosed herein. A CD73 antagonist, shown here as an anti-CD73 antagonist antibody, is added to CD73 expressing cells in combination with AMP. The CD73 antagonist blocks enzymatic conversion of AMP to adenosine. Cells are lysed and any AMP remaining is converted to ADP with a first enzyme (Enzyme I). ADP is then converted to ATP with a second enzyme (Enzyme II), wherein the ATP is used as energy for a luciferase reaction to omit light.

FIGs. 2A-2B shows measurement of AMP of cells incubated in the presence of Triton X- 100 (FIG. 2A) or in the absence of Triton X-100 (FIG. 2B).

FIGs. 3A-3B show a CD73 antagonist potency assay carried out in half-area (FIG. 3A) and full area (FIG. 3B) 96 well plates. 10,000 cells/well and 10 μΜ AMP were used. The assay was carried out 40%, 100% and 160% to detect under potent and hyper potent material. Y axis = relative luminescence units (RLU).

FIGs. 4A-4B show a CD73 antagonist potency assay where a CD73 antagonist (i.e., Ab A) was pre-incubated with SNU-387 cells for 20 minutes (FIG. 4A) or was not pre-incubated (FIG. 4B). The assay was carried out 40%, 100%, 130% and 160% to detect under potent and hyper potent material. Y axis = RLU.

FIG. 5 shows the impact of cell number on the CD73 antagonist potency assay. Various numbers of SNU-387 cells were incubated with 10 μΜ AMP for 1 hour. Representative of two independently run experiments.

FIG. 6 shows the impact of AMP concentration on the CD73 antagonist potency assay. SNU-387 cells (10,000 cells/well) were incubated with a CD73 antagonist (i.e., Ab A) prior to the addition of various concentrations of AMP. The dashed line indicates the AMP-Glo™ reaction behaves linearly from about 1 μΜ to about 100 μΜ AMP. Y axis = RLU.

FIG. 7 shows the impact of AMP-Glo™ reagent volume on the CD73 antagonist potency assay. SNU-387 cells (10,000 cells/well) were incubated with a CD73 antagonist (i.e., Ab A) at the highest (2 μg/mL) and lowest (0.0033 μg/mL) concentrations. Various volumes of AMP- Glo™ reagents (Glo I and Glo II (also referred to as Detection Solution)) were used as indicated in the graph.

FIGs. 8A-8B show the impact of permeabilizing SNU-387 cells with Triton™ X-100 at different concentrations after incubation with a CD73 antagonist (i.e., Ab A) and various concentrations of AMP, wherein permeabilizing occurred without shaking (FIG. 8A) or with shaking (FIG. 8B). FIG. 9 shows the impact of Triton™ X-100 levels on the CD73 antagonist potency assay. SNU-387 cells (10,000 cells/well) were incubated with a CD73 antagonist (i.e., Ab A) at the highest (2 μg/mL) and lowest (0.0033 μg/mL) concentrations.

FIG. 10 shows an initial assessment of Triton X-100 from Electron Microscopy Sciences.

FIGs. 11A-11D show the impact of source of Triton™ X-100 on the CD73 antagonist potency assay. Triton™ X-100 from Life Technologies is shown in FIGs. 11A and 11C and from Electron Microscopy Sciences is shown in FIGs. 11B.

FIGs. 12A-12F show the impact of AMP concentration on the CD73 antagonist potency assay. SNU-387 cells (10,000 cells/well) were incubated with various concentrations of a CD73 antagonist (i.e., Ab A)and concentrations of AMP ranging from 2μΜ to 50 μΜ. Curves are represented together (FIG. 12A) or individually by AMP concentration at 5μΜ (FIG. 12B), 10 μΜ (FIG. 12C), 20μΜ (FIG. 12D), 30μΜ (FIG. 12E) and 50μΜ (FIG. 12F).

FIGs. 13A-13I show the impact of temperature on the CD73 antagonist potency assay. The following temperatures were tested: 16°C (FIG. 13A); 18°C (FIG. 13B); 29°C (FIG. 13C); 37°C (FIG. 13D); 20°C (FIG. 13E); 22°C (FIG. 13F); 24°C (FIG. 13G); 25°C (FIG. 13H); and 27°C (FIG. 131).

FIG. 14A shows CD73 expression levels in SNU-387 cells at passage 18 and passage 30.

FIG. 14B shows performance of the CD73 antagonist potency assay when SNU-387 cells at passage 30 were used.

DETAILED DESCRIPTION

Described herein is a cell-based assay capable of measuring the potency of a CD73 antagonist (a CD73 antagonist potency assay). Specifically, it has been demonstrated that the ability of a CD73 antagonist to inhibit CD73 -mediated enzymatic conversion of AMP to adenosine can be measured in an assay that comprises incubating CD73 expressing cells in the presence of AMP and a CD73 antagonist, and subsequently measuring the amount of remaining AMP, which amount is inversely proportional to the potency of the CD73 antagonist. Thus, the greater the amount of remaining AMP, the more potent the antagonist. As provided herein, a quality control and/or reference sample of a CD73 antagonist having a known potency can be used to determine the potency of a test sample of the CD73 antagonist. Definitions

In order that the present description may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "Cluster of Differentiation 73" or "CD73" as used herein refers to an enzyme (nucleotidase) capable of converting extracellular nucleoside 5’ monophosphates to nucleosides, namely adenosine monophosphate (AMP) to adenosine. CD73 is usually found as a dimer anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) linkage, has ecto- enzyme activity and plays a role in signal transduction. The primary function of CD73 is its conversion of extracellular nucleotides (e.g., 5'- AMP) to adenosine, a highly immunosuppressive molecule. Thus, ecto-5'-nucleotidase catalyzes the dephosphorylation of purine and pyrimidine ribo- and deoxyribonulceoside monophosphates to the corresponding nucleoside. Although CD73 has broad substrate specificity, it prefers purine ribonucleosides.

As used herein, “CD73” refers to human CD73, unless indicated otherwise.

CD73 is also referred to as ecto-5'nuclease (ecto-5'NT, EC 3.1.3.5). The term "CD73" includes any variants or isoforms of CD73 which are naturally expressed by cells. Two isoforms of human CD73 have been identified, both of which share the same N-terminal and C-terminal portions. Isoform 1 (Accession No. NP_002517.1) represents the longest protein, consisting of 574 amino acids and 9 exons. Isoform 2 (Accession No. NP_001191742.1) encodes a shorter protein, consisting of 524 amino acids, lacking amino acids 404-453. Isoform 2 lacks an alternate in-frame exon resulting in a transcript with only 8 exons, but with the same N- and C- terminal sequences.

The terms “CD73 antagonist” of “CD73 inhibitor” are used interchangeably and refer to a biological structure or chemical agent that inhibits, or blocks (partially or completely) the activity of human CD73, i.e., a biological and/or enzymatic function of human CD73. These functions include, for example, the ability of an antibody to inhibit CD73 enzymatic activity, e.g., CD73-regulated production of adenosine or reduction of c AMP production.

Suitable antagonist molecules include, for example, antibodies or fragments thereof (such as the anti-CD73 antibodies described in WO 2016/081748, herein incorporated by reference), small molecules (such as purine derivatives described in US 20170044203, herein incorporated by reference, and those described in US 20170267710, herein incorporated by reference), antisense oligonucleotides, nucleic acid molecules (e.g., siRNA). Molecules capable of inhibiting activity of a protein are known to those of skill in the art. In some embodiments, the CD73 antagonist is an anti-CD73 antibody. In some embodiments, the CD73 antagonist inhibits the activity of human CD73 in a dose-dependent manner. In some embodiments, the activity is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the activity with a negative control under comparable conditions.

The terms “potency” and “activity” are used interchangeably and refer to a measure of the ability of a CD73 antagonist (e.g., an anti-CD73 antibody) to inhibit the activity of human CD73, e.g., the enzymatic activity of human CD73, such as the conversion of AMP to adenosine by CD73.

The term “predefined criterion” refers to one or more values of measurable characteristics of a product. In some embodiments, such values are measurements of the ability of a CD73 antagonist to inhibit enzymatic conversion of AMP to adenosine by CD73. Such values may be used to determine whether a given batch is suitable (e.g., for sale or for medical use), e.g., by comparison to reference values.

The terms “cells expressing human CD73”, “human CD73 expressing cells” and “human CD73+ cells” are used interchangeably and refer to cells that express human CD73 on the surface. In some embodiments, human CD73 is naturally expressed on the cell surface. In some embodiments, cells are engineered to express human CD73 on the cell surface. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells (e.g., murine cells). In some embodiments, the cells are commercially available. In some embodiments, the cells are SNU-387 cells (ATCC Accession No. CVCL_0250).

The term “permeabilizing cells” refers to a process of breaking down the membrane of a cell. Permeabilizing cells is used interchangeably with “lysing cells.” In some embodiments, permeabilizing a cell creates hole in the cell membrane such that intracellular components escape. In some embodiments, permeabilizing a cell destroys the cell membrane such that the entire cell is taken apart. Permeabilizing of cells can occur by viral, enzymatic, osmotic, or physical/mechanical mechanisms, and other methods known to those of skill in the art. In some embodiments, a combination of mechanisms suitable for permeabilizing cells is used. In some embodiments, permeabilizing occurs by contacting the cells with a buffer comprising a molecule capable of breaking down the membrane of a cell. In some embodiments, permeabilizing occurs by contacting the cells with a detergent. In some embodiments, permeabilizing occurs by contacting the cells with a detergent and simultaneously shaking or mixing the cells.

“Shaking” and “mixing” are used interchangeably and refer to a motion that allows for movement of cells suspended in a liquid culture medium. Examples of such motions include, but are not limited to, rotation along a horizontal plane and linear back and forth movements.

The term “detergent” refers to mild surfactants used for cell permeabilization. Detergents are amphiphilic molecules containing both hydrophilic and hydrophobic regions. The amphiphilic property allows detergents to break down protein-protein, protein-lipid and lipid- lipid associations, denature proteins and other macromolecules, and prevent nonspecific binding in immunochemical assays and protein crystallization. There are three main categories of detergents: ionic (sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl), non-ionic (Triton™ X-100, n-dodecyl-B-D-maltoside (DDM), digitonin, Tween® 20, Tween® 80), and zwitterionic (CHAPS). Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be anionic or cationic. Non-ionic detergents have uncharged hydrophilic headgroups. Non-ionic detergents are considered mild surfactants as they do not typically break protein-protein interactions and generally do not denature proteins. Zwitterionic detergents are hydrophilic and contain both positive and negative charges in equal numbers, resulting in zero net charge.

“Enzyme conversion” and “enzyme activity” are used interchangeably and refer to a reaction in which an enzyme acts upon a substrate to generate a product. An enzyme attracts substrates to its active site, catalyzes the chemical reaction by which products are formed, and then allows the products to dissociate. In some embodiments, the methods and assays described herein measure the enzymatic activity of CD73, in which CD73 acts upon AMP (substrate) to generate adenosine (product) by enzymatic conversion. In some embodiments, the methods and assays described herein utilize different enzymatic conversions. For example, in some embodiments an enzyme is used to convert AMP (substrate) to ADP (product). In some embodiments, an enzyme is used to convert ADP (substrate) to ATP (product). In some embodiments, an enzyme is used to convert luciferin (substrate) to oxyluciferin and light (products). “Reference antagonist” refers to a molecule that binds to CD73 and is used to establish a relationship between itself and one or more distinct CD73 antagonists. In some embodiments, the relationship is the activity of the reference antagonist and the one or more district CD73 antagonists. As used herein, the term connotes a CD73 antagonist that is useful in a test or assay, such as those described herein, wherein the assay is useful for the discovery, identification or development of one or more distinct CD73 antagonists that have the same activity. In some embodiments, a reference antagonist has a known potency and is used as a comparator to determine the potency of a sample antagonist.

“Luciferase” refers to an enzyme that catalyzes a chemical reaction that combines an oxygen molecule with a luciferin to form oxyluciferin. “Luciferin” refers to a light-emitting compound found in organisms that generate bioluminescence. Examples of luciferins include, but are not limited to, firefly luciferin and coelenterazine. The reaction releases a luminescent signal (e.g. light) which can be quantified.

“AMP-Glo” and “AMP-Glo™” are used interchangeably and refer to an assay kit manufactured by Promega™. AMP-Glo comprises two reagents: Reagent I which terminates an converts AMP into ADP; and AMP Detection Solution which converts ADP to ATP and uses luciferase to generate a luminescent signal. ATP is the energy source for the oxidation of a luciferin by a luciferase. In some embodiments, AMP-Glo is used to measure the amount of AMP not converted into adenosine by CD73.

A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein may contain a modification such as, but not limited to, glycosylation, phosphorylation or a disulfide bond. A "protein" may comprise one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double- stranded, and may be cDNA.

As used herein, the terms “inhibits” or “blocks” (e.g., referring to inhibition/blocking of CD73 binding or activity) are used interchangeably and encompass both partial and complete inhibition/blocking .

The terms “patient” and “subject” refer to any human or non-human animal that receives either prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat a subject having cancer. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

The term “antibody” as used herein may include whole antibodies and any antigen binding fragments (i.e., “antigen-binding portions”) or single chains thereof. An “antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, CH1, CH2 and CHS. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FRS, CDRS, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The heavy chain of an antibody may or may not contain a terminal lysine (K), or a terminal glycine and lysine (GK). Thus, any of the heavy chain sequences and heavy chain constant region sequences provided herein can end in either GK or G, or lack K or GK, regardless of what the last amino acid of the sequence provides. This is because the terminal lysine and sometimes glycine and lysine are cleaved during expression of the antibody.

Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10^-7 to 10^-11 M or less. Any KD greater than about 10^-6 M is generally considered to indicate nonspecific binding. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a KD of 10^-7 M or less, preferably 10^-8 M or less, even more preferably 5 x 10^-9 M or less, and most preferably between 10^-8 M and 10^-10 M or less, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% or greater sequence identity to the sequence of the given antigen. By way of example, an antibody that binds specifically to human CD73 may also cross-react with CD73 from certain non-human primate species (e.g., cynomolgus monkey), but may not cross-react with CD73 from other species, or with an antigen other than CD73.

An immunoglobulin may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. In certain embodiments, the anti-CD73 antibodies described herein are of the human IgGl or IgG2 subtype. Immunoglobulins , e.g., human IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" may include, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies.

The term “antigen-binding portion” or “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD73). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody, e.g., an anti- CD73 antibody described herein, include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two

Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

The term “Ab A” refers to an antibody or antigen-binding fragment thereof having a variable heavy chain and variable light chain as set forth in SEQ ID NOs: 1 and 2, respectively.

A “bi specific” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs, giving rise to two antigen binding sites with specificity for different antigens. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.

Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope. Typically such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be propagated without intentionally introducing any sequence alterations. Accordingly, the term “human monoclonal antibody” refers to a monoclonal antibody that has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma, for example, obtained by fusing a B cell obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain transgene), to an immortalized cell.

The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen.” An “isolated antibody,” as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CD73 is substantially free of antibodies that specifically bind antigens other than CD73). An isolated antibody that specifically binds to an epitope of CD73 may, however, have cross-reactivity to other CD73 proteins from different species.

As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to binding of an antagonist to an epitope on a predetermined antigen but not to other antigens. Typically, the antagonist (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10^-7 M, such as approximately less than 10 ^-8 M, 10^-9 M or 10^-10 M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BIACORE^® 2000 surface plasmon resonance instrument using the predetermined antigen, e.g., recombinant human CD73, as the analyte and the antagonist as the ligand, or Scatchard analysis of binding of the antagonist to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non- specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely -related antigen. Accordingly, unless otherwise indicated, an antagonist that “specifically binds to human CD73” refers to an antagonist that binds to soluble or cell bound human CD73 with a KD of 10^-7 M or less, such as approximately less than 10 ^-8 M, 10^-9 M or 10^'10 M or even lower.

The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”) In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and maybe a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

As used herein, the term “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. An antigen may be CD73 or a fragment thereof.

The term “CD73 antagonist drug product” refers to a composition comprising a CD73 antagonist that is to be administered to a patient in need of treatment with the CD73 antagonist. In some embodiments, the drug product comprises an anti-CD73 antagonist antibody. In some embodiments, a drug product comprises components in addition to the CD73 antagonist, including those acceptable for pharmaceutical use, e.g., carrier, diluents, adjuvants and excipients.

The terms “passage” and “cell passage”, used interchangeably, refer to a subculture made by transferring some or all cells from a previous culture to a fresh growth medium to allow them to divide to obtain more cells. The number of passages indicates the number of times a cell culture has been subcultured. For example, cells that have been passaged 10 times have been subjected to 10 separate subculturing procedures. Cell passaging enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. In some embodiments, a cell culture is subcultured when the cells are at 90%-100% confluency.

Various aspects described herein are described in further detail in the following subsections. Methods of Measuring CD73 Antagonism

The present disclosure provides methods and in vitro assays for assessing the potency and/or activity of a CD73 antagonist, i.e., the ability of a CD73 antagonist to block, prevent and/or inhibit CD73 -mediated enzymatic conversion of AMP to adenosine. The present disclosure also provides methods and in vitro assays for determining whether a test agent is a CD73 antagonist. Any assay described herein that refers to testing the potency of a CD73 antagonist can also be applied for determining whether a test agent is a CD73 antagonist.

In some embodiments, a method and/or in vitro assay for assessing the potency of a CD73 antagonist comprises the steps of:

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a CD73 antagonist; and

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a CD73 antagonist; and

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a CD73 antagonist; and

In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of: (a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a CD73 antagonist; and

In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73-mediated enzymatic conversion of AMP to adenosine comprises the steps of:

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert

AMP to adenosine in the absence of a CD73 antagonist; and

In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of:

In some embodiments, a method and/or in vitro assay for assessing the activity of a CD73 antagonist comprises the steps of:

(b) measuring the amount of AMP after the period of time as an indication of the activity of the CD73 antagonist.

In some embodiments, a method and/or in vitro assay for assessing the activity of a CD73 antagonist comprises the steps of: (a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a CD73 antagonist; and

In some embodiments, a method and/or in vitro assay for assessing the potency of a test agent as a CD73 antagonist comprises the steps of:

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a test agent; and

(b) measuring the amount of AMP after the period of time as an indication of the potency of the test agent as a CD73 antagonist.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a test agent; and

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a test agent; and (b) measuring the amount of AMP after the period of time as an indication of the potency of the test agent as a CD73 antagonist.

In some embodiments, a method and/or in vitro assay for quantifying the ability of a test agent to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of:

(b) measuring the amount of AMP after the period of time as an indication of the ability of the test agent to prevent enzymatic conversion by CD73.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a test agent; and

In some embodiments, a method and/or in vitro assay for assessing the activity of a test agent as a CD73 antagonist comprises the steps of:

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a test agent; and (b) measuring the amount of AMP after the period of time as an indication of the activity of the test agent as a CD73 antagonist.

(b) measuring the amount of AMP after the period of time as an indication of the activity of the test agent as a CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a CD73 antagonist;

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) permeabilizing the cells, such as with Triton-X100; and (c) measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73.

(b) permeabilizing the cells, such as with Triton-X100; and

In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of: (a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the activity of the CD73 antagonist.

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the activity of the CD73 antagonist. In some embodiments, a method and/or in vitro assay for assessing the potency of a test agent as a CD73 antagonist comprises the steps of:

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of a test agent;

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the potency of the test agent as a CD73 antagonist.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a test agent;

(b) permeabilizing the cells, such as with Triton-X100; and

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of a test agent;

(b) permeabilizing the cells, such as with Triton-X100; and

(b) permeabilizing the cells, such as with Triton-X100; and (c) measuring the amount of AMP after the period of time as an indication of the ability of the test agent to prevent enzymatic conversion by CD73.

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the ability of the test agent to prevent enzymatic conversion by CD73.

(b) permeabilizing the cells, such as with Triton-X100; and

(c) measuring the amount of AMP after the period of time as an indication of the activity of the test agent as a CD73 antagonist.

In some embodiments, a method and/or in vitro assay for assessing the activity of a test agent as a CD73 antagonist comprises the steps of: (a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of a test agent;

(b) permeabilizing the cells, such as with Triton-X100; and

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of the CD73 antagonist;

(b) conducting the step in (a) in the presence of a reference CD73 antagonist, wherein (a) and (b) are conducted simultaneously or sequentially; and

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of the CD73 antagonist;

(b) conducting the step in (a) in the presence of a reference CD73 antagonist, wherein (a) and (b) are conducted simultaneously or sequentially; and (c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the CD73 antagonist;

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73.

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73. In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of:

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the activity of the CD73 antagonist.

In some embodiments, a method and/or in vitro assay for assessing the activity of a CD73 antagonist comprises the steps of: (a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the CD73 antagonist;

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of at least some AMP to adenosine in the absence of the test agent;

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the potency of the test agent as a CD73 antagonist.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of the test agent;

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the test agent; (b) conducting the step in (a) in the presence of a reference CD73 antagonist, wherein (a) and (b) are conducted simultaneously or sequentially; and

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the ability of the test agent to prevent enzymatic conversion by CD73.

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the activity of the test agent as a CD73 antagonist.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the test agent;

(c) comparing the amount of AMP after the period of time in (a) and (b) as an indication of the activity of the test agent as a CD73 antagonist. In some embodiments, a method and/or in vitro assay for assessing the potency of a CD73 antagonist comprises the steps of:

(b) conducting the step in (a) in the presence of a reference CD73 antagonist, wherein (a) and (b) are conducted simultaneously or sequentially;

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the potency of the CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the potency of the CD73 antagonist. In some embodiments, a method and/or in vitro assay for quantifying the ability of a CD73 antagonist to prevent CD73 -mediated enzymatic conversion of AMP to adenosine comprises the steps of:

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and (d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the ability of the CD73 antagonist to prevent enzymatic conversion by CD73.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the activity of the CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and (d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the activity of the CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the potency of the test agent as a CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and (d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the potency of the test agent as a CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the ability of the test agent to prevent enzymatic conversion by CD73.

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the cells to convert AMP to adenosine in the absence of the test agent; and

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the test agent; (b) conducting the step in (a) in the presence of a reference CD73 antagonist, wherein (a) and (b) are conducted simultaneously or sequentially;

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(d) measuring the amount of AMP after the period of time in (a) and (b) and after step (c) as an indication of the activity of the test agent as a CD73 antagonist.

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

(a) contacting cells expressing human CD73 with the test agent and adenosine monophosphate (AMP) for a period of time and under conditions that allow the CD73 expressed on the cells to convert AMP to adenosine in the absence of the test agent; (b) conducting the step in (a) in the presence of a reference CD73 antagonist instead of the test agent, wherein (a) and (b) are conducted simultaneously or sequentially;

(c) permeabilizing the cells in (a) and (b), such as with Triton-X100; and

Steps (a) and (b) e.g., in the above embodiments, may be conducted with different concentrations of test agent and reference CD73 antagonist to create a dose-response curve for the test agent and a dose-response curve for reference CD73 antagonist, e.g., as described in USP 1033 Biological Assay Validation and USP 1034 Analysis of Biological Assays (see, https://www.drugfuture.eom/Pharmacopoeia/usp35/PDF/5186-

5201%20%5bl034%5d%20Analysis%20of%20Biological%20Assays.pdf. A dose-response curve represents the amount of AMP detection signal as a function of the concentration of CD73 antagonist or test agent or reference CD73 antagonist. In certain embodiments, the two curves are compared to determine the activity or potency of the CD73 antagonist or test agent, pursuant to USP 1033 and 1034. The number of different concentrations, e.g., increasing concentrations, of test agent and reference CD73 antagonist for use in the assay is a number that is sufficient to obtain dose-response curves that allow a comparison of the dose-response curve of the test agent and that of the reference CD73 antagonist to be compared for determining the potency of the test agent. In certain embodiments, 4, 5, 6, 7, 8 or more concentrations are tested. In certain embodiments, 8 concentrations are tested. In certain embodiments, the concentrations range from 2 μg/ml to 0.0033μg/mL. In certain embodiments, 8 concentrations from 2 μg/ml to 0.0033μg/mL are tested with each of the test agent and the reference CD73 antagonist.

For example, a method for assessing the potency of a CD73 antagonist (or “test CD73 antagonist”) may comprise the steps of:

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time, wherein the level of AMP is determined by: (i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

A method for assessing the potency of a CD73 antagonist may comprise the steps of:

(a) contacting 4, 5, 6, 7, 8 or more increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

(a) contacting 8 increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

(a) contacting 8 increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

(a) contacting 8 increasing concentrations from 0.0033μg/mL to 2 μg/ml of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(i) permeabilizing the cells with 0.5% Triton X-100; (ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

(a) contacting 8 increasing concentrations from 0.0033μg/mL to 2 μg/ml of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

(c) comparing the two dose-response curves to thereby assess the potency of the CD73 antagonist. In certain embodiments, an assay includes a quality control CD73 antagonist, which is used to ensure that the assay is performing as intended. A quality control CD73 antagonist may be used as a first step to confirm that the system is performing correctly, and after determining that it is performing correctly, the actual activity of the test sample is determined by comparison with the reference material, e.g., CD73 antagonist. A quality control CD73 antagonist can be anything that you can use to ensure that the test system is behaving normally. For example, it can be 1) a separate preparation of reference standard, 2) a different batch/lot of the same antagonist as that of the CD73 antagonist or test sample, or even 3) a different antagonist (eg. biosimilar or other antagonist).

In some embodiments, the methods described herein are useful for analyzing different batches of manufactured CD73 antagonist. Accordingly, the present disclosure also provides a method and/or in vitro assay for determining whether a manufactured CD73 antagonist fulfills a predefined criterion (e.g., has a potency that is the same as that of a reference standard CD73 antagonist), the method comprising:

(a) contacting cells expressing human CD73 with the CD73 antagonist and adenosine monophosphate (AMP) for a period of time and under conditions that allow the conversion of AMP to adenosine in the absence of a CD73 antagonist; and

(b) measuring the amount of AMP after the period of time.

In some embodiments, the inhibition of CD73 -mediated enzymatic conversion of AMP to adenosine by a CD73 antagonist is compared to the inhibition by a reference antagonist, and the therapeutic efficacy of the CD73 antagonist is assessed from its ability to inhibit CD73 -mediated enzymatic conversion to the same or substantially the same degree as the reference antagonist.

In some embodiments, the inhibition of CD73-mediated enzymatic conversion of AMP to adenosine by a reference standard is a suitable indicator for the potency of the reference standard, and any CD73 antagonist showing the same or substantially the same inhibition as the reference standard is deemed to have the same or substantially the same therapeutic activity as the reference standard. In some embodiments, the degree to which inhibition by the CD73 antagonist and inhibition by the reference standard may differ is established on a case-to-case basis.

To determine inhibition of CD73 -mediated enzymatic conversion of AMP to adenosine in a reliable and consistent manner, in some embodiments the inhibition by a CD73 antagonist and the reference standard are performed using the same assay. In some embodiments, the inhibition by the reference standard is performed first to establish a standard that any following batches can be compared with.

In some embodiments, the disclosure provides a method of producing a pharmaceutical composition comprising a CD73 antagonist, the method comprising:

(a) the production of a drug product comprising a CD73 antagonist;

(b) subjecting said drug product to a method as described herein for determining the potency of the CD73 antagonist; and

(c) using the information obtained in step (b) as part of an assessment of whether the drug product may be used as a pharmaceutical composition. The production of a drug product may be performed in any manner as desired and/or suitable for the drug product in question. In some embodiments, the drug product is subjected to a method for assaying the inhibition of CD73 -mediated enzymatic conversion of AMP to adenosine. In some embodiments, the result of this method is an indication as to whether the drug product may be used as a pharmaceutical composition. In some embodiments, a key feature in the production of the drug product is to ensure that different batches live up to the same standard. In some embodiments, the standard is set in cooperation with a regulatory body.

Assay Components

The methods and assays described herein utilize at least CD73 expressing cells, a CD73 antagonist (e.g., for testing and/or as a reference standard/quality control), and AMP. In some embodiments, the methods and assays further utilize an agent, such as a detergent to permeabilize the CD73 expressing cells. In some embodiments, the methods and assays further utilize any methods and reagents suitable for measuring the amount of AMP.

CD73 Expressing Cells

CD73 expressing cells for use in the methods, assays and kits described herein may be any cells expressing human CD73 or a portion thereof, whether endogenously or exogeneously expressing CD73, that provide the expected results in the assays described herein with a reference CD73 antagonist, i.e., a CD73 antagonist with known enzymatic activity.

CD73 can be endogenous or exogenous to a cell. The term “endogenous CD73” refers to a CD73 polypeptide naturally expressed by the cell because it is naturally encoded within the cell’s genome, such that the cell inherently expresses CD73 without the need of an external source of CD73 or an external source of genetic material encoding CD73. Expression of endogenous CD73 may be with or without environmental stimulation such as e.g., cell differentiation or promoter activation. The term “exogenous CD73” refers to a CD73 polypeptide expressed in a cell through the introduction of an external source of CD73 or an external source of genetic material encoding CD73 by human manipulation. The expression of exogenous CD73 may be with or without environmental stimulation such as e.g., cell differentiation or promoter activation. In some embodiments, cells from an established cell line can express one or more exogenous CD73 polypeptides by transient or stable transfection of a polynucleotide molecule encoding a CD73 polypeptide. In some embodiments, cells from an established cell line can express one or more exogenous CD73 polypeptides by protein transfection of the CD73. An exogenous CD73 polypeptide can be naturally occurring CD73 or naturally occurring variants thereof, or non-naturally occurring CD73 or non-naturally occurring variants thereof.

In some embodiments, cells useful in the methods and assays provided herein endogenously express CD73. In some embodiments, cells for use in the methods and assays provided herein are cells genetically engineered to express CD73. In some embodiments, cells for use in the methods and assays provided herein are human cells endogenously expressing human CD73. In some embodiments, cells for use in the methods and assays provided herein are human cells genetically engineered to express human CD73. In some embodiments, cells for use in the methods and assays provided herein are human cells genetically engineered to express non-human CD73. In some embodiments, cells for use in the methods and assays provided herein are non-human cells genetically engineered to express human CD73. In some embodiments, cells for use in the methods and assays provided herein are non-human cells genetically engineered to express non-human CD73.

Methods for genetically engineering cells to express surface proteins are known to those of skill in the art. For example, in some embodiments a cell is transfected with a plasmid comprising a nucleic acid encoding a CD73 protein. In some embodiments, host cells are contacted with a vector comprising a nucleic acid encoding a CD73 protein.

The number of times a cell culture is passaged can influence cell viability along with the amount of CD73 expressed on the surface of the cells. Methods for determining cell viability and CD73 expression on a cell is known to those of skill in the art and are described infra. Accordingly, in some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged between 0-50 times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged between 0-40 times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged between 0-30 times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged between 0-20 times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged between 0-10 times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged 50 or less times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged 40 or less times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged 30 or less times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged 20 or less times. In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein have been passaged 10 or less times. Generally, cells for use in assays for determining CD73 enzymatic activity described herein express a sufficient level of CD73 on the cell surface to allow the assay to function appropriately.

In some embodiments, CD73 expressing cells suitable for use in the methods and assays described herein are capable of converting soluble AMP to adenosine.

In some embodiments, the CD73 antagonists, e.g., those described herein and those known to those of skill in the art, are capable of binding, e.g., specifically binding, to CD73 expressed on the surface of the cells.

Exemplary CD73 expressing cells for use in the methods, assays and kits described herein are SNU-387 cells (ATCC Accession No. CRL-2237) or Calu-6 cells (ATCC Accession No. HTB-56). In some embodiments, CD73 expressing cells for use in the methods and assays described herein are SNU-387 cells. SNU-387 cells have from 100,000 to 200,000 CD73 molecules on their cell surface. In some embodiments, CD73 expressing cells used in the methods, assays and kits described herein are Calu-6 cells (human pulmonary adenocarcinoma cell line). In certain embodiments, SNU-387 cells are cells from passage 3 up to and including passage 30 (see Examples). The acceptable number of passages for any cell line can be determined by conducting the assay described herein with cells at different passages, and determining which cells (at which passages) are able of providing the expected CD73 antagonist activity of a reference CD73 antagonist.

Other human CD73 positive cell lines that may be used include the following: H2228 (human non-small cell lung cacinoma cell line), SKLU1 (human lung adenocarcinoma), HCC15 (human non-small cell lung cacinoma cell line), HCC44 (human non- small cell lung cacinoma cell line), H647 (human non- small cell lung cacinoma cell line), H2030 (human non- small cell lung cacinoma cell line), NCI-292; SNU-C1 (human colon cancer cell line), NCI-H1437 (human non- small cell lung cacinoma cell line), SKMES 1 (human melanoma cell line), SW900 (human squamous cell lung carcinoma), SK-MEL-24 (human melanoma cell line). These are all cell lines that have been shown to bind Ab A (see WO 2016/081748). A CD73 negative cell line that may be used as a negative control include DMS 114 (human small cell lung cancer cell line).

The number of CD73 molecules on the surface of the cells (i.e., the CD73 cell surface density) should be at least a number that is sufficient for providing the expected results in the assays described herein with a reference CD73 antagonist, i.e., a CD73 antagonist with known activity. In some embodiments, cells (e.g., a cell line) for use in the method, assay and kits described herein comprises a range from 100,000 to 200,000 CD73 molecules per cell. In some embodiments, the cells comprise a number of cell surface CD73 molecules that is in the same range as that of SNU-387 or Calu-6 cells, e.g., SNU-387 cells from passages 3 to 30. The number of CD73 molecules on the cell surface can be determined according to methods known in the art. For example, density of surface molecules can be determined by saturation binding of a soluble ligand.

Cells that express endogenous or exogenous CD73 can be identified by routine methods. Assays that determine CD73 binding can be used to assess whether a cell is expressing CD73. Non-limiting assays include immunocytochemical assays that detect CD73 using labeled or unlabeled antibodies, and immunoprecipitation assays. Where an antibody useful in these assays is labeled, the binding of the molecule can be detected by various means, including Western blot analysis, direct microscopic observation of the cellular location of the antibody, measurement of cell or substrate-bound antibody, flow cytometry, electrophoresis or capillary electrophoresis. If the antibody is unlabeled, a labeled secondary antibody can be used for indirect detection of the bound molecule, and detection can proceed as for a labeled antibody.

In some embodiments, 5,000-30,000 cells/well of a 96-well plate are used. In some embodiments, 10,000-25,000 cells/well of a 96-well plate are used. In some embodiments, 15,000-20,000 cells/well of a 96-well plate are used. In some embodiments, about 5,000 cells/well of a 96-well plate are used. In some embodiments, about 10,000 cells/well of a 96- well plate are used. In some embodiments, about 15,000 cells/well of a 96-well plate are used.

In some embodiments, about 20,000 cells/well of a 96-well plate are used. In some embodiments, about 25,000 cells/well of a 96-well plate are used. In some embodiments, about 30,000 cells/well of a 96-well plate are used. Cell numbers outside of the 5,000-30,000 range may also be used, but these might require adjusting several parameters of the assay, such as AMP levels and incubation times. Smaller or larger wells may also be used (e.g., 384 well or 48 well plates) may be used by adjusting the number of cells respective to the cell numbers used for wells of a 96 well plate set forth above.

AMP

To measure the ability of a CD73 antagonist to inhibit CD73 -mediated enzymatic conversion of AMP to adenosine or to determine whether a test agent inhibits CD73 mediated conversion of AMP to adenosine, the methods, assays and kits described herein utilize exogenous AMP. Dephosphorylation of AMP generates adenosine. As described supra, CD73 is an enzyme that catalyzes the dephosphorylation of AMP to generate adenosine.

In some embodiments, AMP is added after CD73 expressing cells have been contacted with a CD73 antagonist. For example, an assay for determining the potency of a CD73 antagonist or for determining if a test agent is a CD73 antagonist, may comprise first combining CD73 positive cells and the and-CD73 antagonist or test agent, respectively, mixing the cells and the antagonist or test agent, respectively, and incubating the mixture for 0 to 25 minutes, e.g., at room temperature, prior to adding AMP. In certain embodiments, the cells and the antagonist or test agent are incubated together up to 5, 10, 15 or 20 minutes prior to adding AMP. In some embodiments, AMP is added to CD73 expressing cells before contact with a CD73 antagonist or test agent. In some embodiments, AMP and a CD73 antagonist or test agent are added simultaneously or at about the same time to CD73 expressing cells.

In some embodiments, the AMP concentration used in the methods and assay described herein is 10μΜ to 100μM. In certain embodiments, the AMP concentration is 15 to 100μΜ, 20 to 100μΜ or 25 to 100μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is about 10μΜ, 20μΜ, 25μΜ, 30μΜ, 40μΜ, 50μΜ, 60μΜ, 70μΜ, 80μΜ, 90μΜ, 100μΜ, 110μΜ, 120μΜ, 130μΜ, 140μΜ or 150μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 10μΜ or about 10μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 15μΜ or about 15μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 20μΜ or about 20μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 25 μΜ or about 25μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 30μΜ or about 30μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 35μΜ or about 35 μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 40μΜ or about 40μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 45 μΜ or about 45μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 50μΜ or about 50μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 55μΜ or about 55μ M. In some embodiments, the AMP concentration used in the methods and assay described herein is 60μΜ or about 60μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 65 μΜ or about 65μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 70μΜ or about 70μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 75μΜ or about 75 μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 80μΜ or about 80μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 85 μΜ or about 85μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 90μΜ or about 90 μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 95μΜ or about 95 μΜ. In some embodiments, the AMP concentration used in the methods and assay described herein is 100μΜ or about 100μΜ.

AMP from any reliable source may be used in the assays described herein. In one embodiment, AMP from the Promega AMP-Glo™ kit is used.

In the methods described herein, once the cells, CD73 antagonist (or test agent) and AMP are combined in a vial or in a well of a multiwell plate, these ingredients are mixed together, e.g., by shaking of a plate shaker, for a time sufficient to properly mix the ingredients, e.g., from 30 seconds to one or two minutes. For example, shaking may be conducted from 30 seconds to 120 seconds, from 30 seconds to 90 seconds or from 30 seconds to 60 seconds. Shaking may be conducted at room temperature. Shaking may be conducted protected from light. Shaking may be conducted at room temperature, such as at a temperature ranging from 20 to 27°C, such as 20 to 25 °C. In certain embodiment, the combination of CD73 positive cells, antagonist (or test agent) and AMP are combined and mixed together at room temperature and protected from the light. Following mixing of the cells, antagonist and AMP, this reaction mixture is incubated for a time sufficient for allowing the conversion of an acceptable amount, e.g., about 50%, of the added AMP into adenosine. A dose response curve is generally optimized around the EC₅₀ (prevention of conversion of about 50% of the AMP). In some embodiments, the reaction is conducted for 50 to 70 minutes, e.g., 60 minutes. Incubation is conducted at ambient temperature, e.g., from 20 to 27 °C. The incubation may be conducted protected from light. In certain embodiment, the combination of CD73 positive cells, antagonist (or test agent) and AMP are combined and mixed together, and incubated at room temperature and protected from the light for the conversion of AMP to adenosine to occur.

CD73 Antagonists

The methods and assays described herein are useful for determining the ability of a molecule or product to inhibit the conversion of AMP into adenosine, such as to inhibit the enzymatic activity of CD73, and for determining or measuring the potency and/or activity of a molecule that inhibits the conversion of AMP into adenosine, such as that of a CD73 antagonist. CD73 antagonists inhibit, block or prevent CD73 -mediated enzymatic conversion of AMP to adensonine. In some embodiments, the potency and/or activity of a CD73 antagonist is determined by comparison to a CD73 antagonist with a known potency and/or activity (i.e., a reference CD73 antagonist or quality control CD73 antagonist). In some embodiments, a reference or quality control CD73 antagonist is used to generate a response curve based on serial dilutions of the CD73 antagonist or test agent. The response curve is then used to determine the potency and/or activity of the CD73 antagonist being tested.

A reference CD73 antagonist can be any CD73 antagonist having a known potency. The potency of a CD73 antagonist is defined by criteria including its affinity for CD73, its binding kinetics and its binding site. In certain embodiments, a reference CD73 antagonist is Ab A produced in a given manufacturing batch. Every CD73 antagonist that is manufactured has its own reference standard, and such reference standards can be used for determining the potency of another CD73 antagonist or another batch of the same CD73 antagonist as the reference standard.

A CD73 antagonist is any molecule that reduces or inhibits the conversion of AMP to adenosine by CD73 (CD73 enzymatic activity), and can be a molecule, e.g., a protein, that binds to CD73 and thereby inhibits its enzymatic activity, such as an antibody, or an antigen binding fragment thereof, a bispecific antibody, an scFv or other antibody variant, such as those described herein. A CD73 antagonist can also be an alternative scaffold molecule, such as a 10Fn3 (e.g., adnectin) molecule.

Anti-CD73 Antibodies

In some embodiments, the activity and/or potency of an anti-CD73 antagonist antibody, or antigen binding fragment thereof, is measured with the methods and assays described herein.

In some embodiments, anti-CD73 antibodies specifically bind human CD73. In some embodiments, anti-CD73 antibodies cross react with CD73 from one or more non-human primates, such as cynomolgus CD73. In some embodiments, an anti-CD73 antibody binds to soluble CD73. In some embodiments, an anti-CD73 antibody binds to membrane bound CD73. In some embodiments, an anti-CD73 antibody binds to soluble and membrane bound CD73. In some embodiments, anti-CD73 antibodies compete for binding to CD73 with other CD73 antagonists (e.g., other anti-CD73 antibodies).

Standard assays to evaluate the binding ability of the antibodies toward CD73 of various species are known in the art, including for example, ELIS As, Western blots, and RIAs. The binding kinetics (e.g. , binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by BIACORE® SPR analysis.

In some embodiments, anti-CD73 antibodies are not native antibodies or are not naturally-occurring antibodies. For example, in some embodiments anti-CD73 antibodies have post-translational modifications that are different from those of antibodies that are naturally occurring, such as by having more, less or a different type of post-translational modification, e.g., glycosylation.

In some embodiments, the anti-CD73 antibody is one described in WO 2016/081748 (herein incorporated by reference), e.g., having heavy and light chain variable amino acid sequences set forth in SEQ ID NOs: 135 and 12, respectively).

In some embodiments, the anti-CD73 antibody is CPI-006 or CPX-006 (Corvus Pharmaceuticals), described in WO2017/152085 (herein incorporated by reference). In some embodiments, the anti-CD73 antibody is GS-1423 (Agenus, licensed to Gilead), described in US 20190352418 (herein incorporated by reference). In some embodiments, the anti-CD73 antibody is MEDI9447 (oleclumab; Medimmune) or Phen 0203hIgGl, as described in W02016/075099 (herein incorporated by reference). In some embodiments, the anti-CD73 antibody is NZV930 (Novartis and Surface Oncology), described in US 20190031766 (herein incorporated by reference). In some embodiments, the anti-CD73 antibody is TJ004309, also known as TJD5 (Tracon Pharmaceuticals). In some embodiments, the anti-CD73 is an antibody described in W02016/055609 or WO2017/152085, each of which is herein incorporated by reference.

Small Molecules

In some embodiments, the activity and/or potency of a small molecule that inhibits, blocks or prevents CD73 -mediated enzymatic conversion of AMP to adenosine can be measured with the methods and assays described herein. Small molecules that act as CD73 antagonists are known to those of skill in the art.

Methods for determining whether a small molecule binds to CD73 and/or antagonizes CD73 function are known to those of skill in the art and described herein.

Exemplary small molecules include, but are not limited to, those described in US 20170044203 and US 20170267710, each of which is herein incorporated by reference. In some embodiments, the small molecule is LY3475070 (Eli Lilly and Company; NCT04148937).

Intracellular agents

The assay provided herein may also be used to determine the activity of molecules that inhibit the expression of an enzyme that converts AMP to adenosine, such as CD73. Exemplary molecules that inhibit the expression of CD73 include molecules that act intracellularly, such as siRNAs.

Cell Permeabilization Agents

As described herein, CD73 expressing cells are contacted with AMP under conditions that allow for the enzymatic conversion of AMP to adenosine. The ability of a CD73 antagonist to inhibit this CD73 -mediated enzymatic conversion is determined based on the amount of AMP remaining after the reaction, which is inversely correlated with the enzymatic activity of the CD73 antagonist or test agent. To determine the amount of AMP remaining after the reaction, an agent that permeabilizes the cells (or punctures the cell membrane) is added to the reaction mixture, after the reaction.

Exemplary agents that permeabilize cells or puncture the cells (“cell permeabilization agents”) that can be used in the assays described herein are those that are used for detecting intracellular proteins or markers in cells during flow cytometry. In certain embodiments, the cell permeabilization agent is a detergent, such as a non-ioinic detergent, such as Triton X-100.

In some embodiments, the detergent suitable for the methods and assays described herein is a nonionic detergent, such as a mild non-ioinic detergent. Non-ionic detergents have uncharged hydrophilic headgroups and are considered mild surfactants as they do not typically break protein-protein interactions and generally do not denature proteins. Exemplary nonionic detergents include, but are not limited to Triton™ X-100, n-dodecyl-B-D-maltoside (DDM), digitonin, saponin, Tween® 20, Tween® 80 and pore-forming toxins, such as alpha-toxin and streptolysin O.

It may be possible to use electropermeabilization to permeabilize the cells. It may also be possible to use a zwitterionic detergent to permeabilize the cells. Zwitterionic detergents are hydrophilic and contain both positive and negative charges in equal numbers, resulting in zero net charge. An exemplary zwitterionic detergent includes, but is not limited to, CHAPS.

It may also be possible to use an ionic detergent to permeabilize the cells. Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be anionic or cationic. Exemplary ionic detergents include, but are not limited to sodium dodecyl sulfate (SDS), deoxycholate, cholate and sarkosyl.

The amount of detergent added to the reaction is an amount that is sufficient for permeabilizing the cells sufficiently so that a correct reading of the remaining AMP after the reaction can be obtained. In some embodiments, , the final concentration of the detergent in the reaction mixture is about 0.05% to 0.5%, such as about 0.05% to 0.4%, 0.05% to 0.3%, 0.05% to 0.2% or 0.05% to 0.1%. In certain embodiments, 25 μL of a detergent stock solution of about 0.2% to 2% is added to a reaction mixture of 75 μL comprising the cells, the antagonist and AMP. A wide range of concentrations of detergent will achieve its purpose of permeabilizing the cells, however, it is preferably to use the lowest concentration possible, such as to limit the amount of detergent added, while retaining sufficient permeabilization conditions.

Permeabilization of the cells may be conducted for a time period ranging from 3 to 10 minutes, such as about 5 minutes. The permeabilization step may be conducted at room temperature, e.g., a temperature of 20 to 27 °C, such as 20 to 25 °C. The permeabilization step may be conducted in the absence of light (e.g., the reaction mixture is protected from light).

In some embodiments, cells are subjected to shaking while in contact with a detergent. Shaking includes moving cells in a way that keeps them from being stationary. As shown in the Examples, while permeabilization appeared to occur whether or not mixing was present, only the shaking condition yielded a saturating condition. Examples of such motions include, but are not limited to, rotation along a horizontal plane and linear back and forth movements. In some embodiments, a cell culture vessel or container (e.g., cell culture plate) is placed on a shaker that subjects the cells to such a motion. In some embodiments, the reaction mixture comprising the cells, the antagonist, AMP and the detergent are mixed for 5 minutes on a plate shaker set to about 300RPM. In certain embodiments, the mixing is protected from light.

In some embodiments, the detergent suitable for the methods and assays described herein is a Triton detergent, such as Triton X-100, Triton X-114, Nonidet P-40 (NP-40), and Igepal® CA-630. Members of the Triton family are quite similar, differing slightly in their average number (n) of monomers per micelle (9.6, 8.0, 9.0, and 9.5, respectively) and the size distribution of their polyethylene glycol (PEG)-based headgroup. Triton X-100, a typical non- ionic detergent, derives from polyoxyethylene and contains an alkylphenyl hydrophobic group.

In some embodiments, the detergent suitable for the methods and assays described herein is Triton X-100. In some embodiments, the purity and/or quality of Triton X-100 is the same or substantially similar to Triton X-100 manufactured by Life Technologies/Thermo Fisher Scientific (see Examples), which, e.g., has not aged or been significantly degraded. Methods for comparing the purity and/or quality of a detergent, such as Triton X-100 to that of the Triton X- 100 of Life Technologies/ThermoFisher are described in Example 11. Generally, a detergent, such as Triton X-100, is suitable for the assays and methods described herein if the assay using it has no significant inter-plate or intra-plate replicate result variability; there is no significant variability between identical samples of the reference standard; the dose-response curve of the CD73 antagonist reference standard and that of the test sample (or CD73 antagonist) have parallel asymptotes; the dose response curve is sigmoidal; and/or the values fit into a 4-parameter logistic curve. Thus, the use of an unacceptable detergent, such as an unfit batch of Triton X- 100, in the assays and methods described herein would result in increased variability among samples, and/or losing the sigmoidal dose response, the 4 parameter fit, and/or the curve parallelism.

In some embodiments, the concentration of Triton X-100 used in the methods and assays described herein is about 0.2% to about 2.0%. In some embodiments, the Triton X-100 concentration is about 0.2% to about 1.5%. In some embodiments, the Triton X-100 concentration is about 0.2% to about 1.0%. In some embodiments, the Triton X-100 concentration is about 0.2% to about 0.5%. In some embodiments, the Triton X-100 concentration is 0.2%, 0.3%, 0.4%, 0.5%. 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%. In some embodiments, the Triton X-100 concentration is about 0.5%. In some embodiments, the Triton X-100 concentration is about 1%.

Reagents for measuring AMP levels

In some embodiments, the amount of AMP present in the reaction after the permeabilization step indicates the potency and/or activity of a CD73 antagonist. In certain embodiments, the amount of AMP is determined by converting AMP to ATP and measuring the level of ATP, e.g., with a system that uses ATP to emit light, wherein the amount of light is then detected In some embodiments, the method for measuring the amount of AMP is described in WO 2013/063563, herein incorporated by preference.

In some embodiments, the amount of ATP is determined by use of a lucif erase enzyme and a luciferin substrate, in which ATP is an energy source for the oxidation of luciferin resulting in emission of light. The emission of light can be measured using a luminometer.

In some embodiments, the AMP present in the reaction mixture after the permeabilization step is converted into ADP with a first enzyme, which ADP is then converted into ATP with a second enzyme. In certain embodiments, the first enzyme is capable of converting at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the AMP in the reaction mixture to ADP. In some embodiments, the first enzyme is polyphosphate:AMP phosphotransferase (PAP). In some embodiments, polyphosphate is added along with the PAP.

In some embodiments, the second enzyme, which converts ADP to ATP, is capable of converting at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the ADP in the reaction mixture to ATP. In certain embodiments, the second enzyme is adenylate kinase (AK).

In some embodiments, the level of AMP present after the permeabilization step is measured using the AMP-Glo™ Assay kit from Promega (Catalog No. V5012). Specifically, in some embodiments, AMP-Glo™ Reagent I is the first enzyme, and AMP-Glo™ Detection Solution is the second enzyme. The Detection Solution also comprises luciferase and luciferin for measuring levels of ATP.

In some embodiments, the first enzyme: second enzyme ratio is 1:2. In some embodiments, the Reagent Detection Solution volume ratio is 1:2.

In some embodiments, the volume of Reagent I added to the reaction mixture after the permeabilization step, where the reaction mixture has a volume of about 100μL is about 20μL, about 25μL, about 30 μL, about 35μL, about 40μL, about 45 μL, or about 50μL. In some embodiments, the volume of Detection Solution used is about 40μL, about 45 μL, about 50μL, about 55 μL, about 60μL, about 65 μL, about 70μL, about 75 μL, about 80μL, about 85 μL, about 90μL, about 95 μL, or about 100μL. In some embodiments the volume of Reagent I is about 20μL and the volume of Detection solution is about 40μL. In some embodiments the volume of Reagent I is about 25 μL and the volume of Detection solution is about 50μL. In some embodiments the volume of Reagent I is about 30μL and the volume of Detection solution is about 60μL. In some embodiments the volume of Reagent I is about 35 μL and the volume of Detection solution is about 70μL. In some embodiments the volume of Reagent I is about 40 μL and the volume of Detection solution is about 80μL. In some embodiments the volume of Reagent I is about 45 μL and the volume of Detection solution is about 90μL. In some embodiments the volume of Reagent I is about 50μL and the volume of Detection solution is about 100μL. In certain embodiments, the ratio of Reagent I: Detection solution is 1:2. In some embodiments, the volume of Reagent I added to the reaction mixture is 30μL to 40μL and the volume of Detection solution is 60μL to 80μL.

Assay Conditions

In some embodiments, the temperature at which each of the steps of the methods described herein are conducted is room temperature. In some embodiments, the temperature is ambient temperature. In some embodiments, the methods and assays described herein are performed at 20°C - 27°C. In some embodiments, the methods and assays described herein are performed at 21°C - 26°C. In some embodiments, the methods and assays described herein are performed at 22°C - 25°C. In some embodiments, the methods and assays described herein are performed at 23°C - 24°C.

Calculation of the potency of the CD73 antagonist

Using the results obtained in the assays described herein, it is within the skill in the art to derive the potency of a CD73 antagonist or test agent. For example, methods described in USP 1033 Biological Assay Validation and USP 1034 Analysis of Biological Assays (see, https://www.drugfuture.eom/Pharmacopoeia/usp35/PDF/5186-

5201%20%5bl034%5d%20Analysis%20of%20Biological%20Assays.pdf) can be used. The protocols and methods described therein are specifically incorporated by reference herein.

Exemplary Methods and Assays

In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises:

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow the conversion of AMP to adenosine in the absence of a CD73 antagonist; and

(a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist and AMP, for a period of time and under conditions that allow the CD73 of the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) measuring the amount of AMP in the first and second populations of cells after the period of time; and (c) comparing the amount of AMP determined in (b) to derive the potency of the CD73 antagonist.

(a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73, and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) measuring the amount of AMP in the cells after the period of time; and

(c) comparing the amount of AMP determined in (b) to derive the potency of the CD73 antagonist.

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time;

(c) measuring the amount of AMP in the cells; and

(d) comparing the two dose response curves to thereby assess the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow the conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) adding a detergent to the cells after the period of time; and

(c) measuring the amount of AMP as an indication of the potency of the CD73 antagonist. In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises:

(b) adding a detergent to the first and second populations of cells after the period of time;

(c) measuring the amount of AMP in the first and second populations of cells; and

(d) comparing the amount of AMP determined in (c) to derive the potency of the CD73 antagonist.

(b) adding a detergent to the cells after the period of time;

(c) measuring the amount of AMP in the cells; and

(d) comparing the amount of AMP measured between the reference standard CD73 antagonist and the CD73 antagonist.

(c) adding a detergent to the cells after the period of time;

(d) measuring the amount of AMP in the cells; and (e) comparing the two dose response curves to thereby assess the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow the CD73 of the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) adding Triton™ X-100 to the cells after the period of time; and

(c) measuring the amount of AMP as an indication of the potency of the CD73 antagonist.

(a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist having a known potency and AMP, for a period of time and under conditions that allow the CD73 of the cells to convert AMP to adenosine in the absence of a CD73 antagonist;

(b) adding Triton™ X-100 to the first and second populations of cells after the period of time;

(c) measuring the amount of AMP in the first and second populations of cells as an indication of potency; and

(d) comparing the amount of AMP determined by (c) to derive the potency of the CD73 antagonist.

(b) adding Triton™ X-100 to the cells after the period of time;

(c) measuring the amount of AMP in the cells; and (d) comparing the amount of AMP measured between the reference standard CD73 antagonist and the CD73 antagonist.

(c) adding Triton™ X-100 to the cells after the period of time;

(d) measuring the amount of AMP in the cells; and

(e) comparing the two dose response curves to thereby assess the potency of the CD73 antagonist.

(b) adding a detergent to the cells after the period of time and shaking the cells; and

(a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist and AMP, for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) adding a detergent to the first and second populations of cells after the period of time and shaking the cells; (c) measuring the amount of AMP in the first and second populations of cells; and

(b) adding a detergent to the cells after the period of time and shaking the cells;

(c) measuring the amount of AMP in the cells; and

(c) adding a detergent to the cells after the period of time and shaking the cells;

(d) measuring the amount of AMP in the cells; and

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist; (b) adding Triton™ X-100 to the cells after the period of time and shaking the cells; and

(b) adding Triton™ X-100 to the first and second populations of cells after the period of time and shaking the cells;

(b) adding Triton™ X-100 to the cells after the period of time and shaking the cells;

(c) measuring the amount of AMP in the cells; and

(a) contacting 4, 5, 6, 7, 8 or more increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist; (b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time;

(c) adding Triton™ X-100 to the cells after the period of time and shaking the cells;

(d) measuring the amount of AMP in the cells; and

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist; and

(b) measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist by:

(i) adding a first reagent comprising a first enzyme capable of converting AMP to

ADP;

(ii) adding a second reagent comprising a second enzyme capable of converting ADP to ATP; and

(iii) determining the amount of ATP as an indication of the amount of AMP.

(b) measuring the amount of AMP in the first and second populations of cells after the period of time by:

ADP;

(ii) adding a second reagent comprising a second enzyme capable of converting ADP to ATP; and (iii) determining the amount of ATP as an indication of the amount of AMP,

(b) measuring the amount of AMP in the cells after the period of time by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time, wherein the level of AMP is determined by;

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP, (c) comparing the two dose response curves to thereby assess the potency of the CD73 antagonist.

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) adding a detergent to the cells after the period of time; and

(c) measuring the amount AMP as an indication of the potency of the CD73 antagonist by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP.

(c) measuring the amount of AMP in the first and second populations of cells by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(d) comparing the amount of AMP determined in (c) to derive the potency of the CD73 antagonist. In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises:

(b) adding a detergent to the cells after the period of time;

(c) measuring the amount of AMP in the cells by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(i) permeabilizing the cells with a detergent;

(ii) adding a first reagent comprising a first enzyme capable of converting AMP to ADP to the cells of (i);

(iii) adding a second reagent comprising a second enzyme capable of converting

ADP to ATP to the cell of (ii); and

(iv) determining the amount of ATP as an indication of the amount of AMP, (c) comparing the two dose response curves to thereby assess the potency of the CD73 antagonist.

(b) adding Triton™ X-100 to the cells after the period of time; and

(c) measuring the amount of AMP as an indication of the potency of the CD73 antagonist by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP.

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(d) comparing the amount of determined in (c) to derive the potency of the CD73 antagonist. In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises:

(b) adding Triton™ X-100 to the cells after the period of time;

(c) measuring the amount of AMP in the cells by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(d) comparing the amount of determined in (c) to derive the potency of the CD73 antagonist.

(i) permeabilizing the cells with Triton X-100;

(iii) adding a second reagent comprising a second enzyme capable of converting

ADP to ATP to the cell of (ii); and

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP.

(b) adding a detergent to the first and second populations of cells after the period of time and shaking the cells;

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(c) measuring the amount of AMP in the cells by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(i) permeabilizing the cells with a detergent and shaking the cells;

(iii) adding a second reagent comprising a second enzyme capable of converting

ADP to ATP to the cell of (ii); and

(b) adding Triton™ X-100 to the cells after the period of time and shaking the cells; and

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP.

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(d) comparing the amount of AMP determined in (d) to derive the potency of the CD73 antagonist. In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises:

(c) measuring the amount of AMP in the cells by:

ADP;

(iii) determining the amount of ATP as an indication of the amount of AMP,

(d) comparing the amount of AMP determined in (d) to derive the potency of the CD73 antagonist.

(i) permeabilizing the cells with Triton X-100 and shaking the cells;

(iii) adding a second reagent comprising a second enzyme capable of converting

ADP to ATP to the cell of (ii); and

(a) contacting cells expressing human CD73 with the CD73 antagonist and AMP for a period of time and under conditions that conversion of AMP to adenosine in the absence of a CD73 antagonist; and

(b) measuring the amount of AMP with an AMP-Glo™ Assay after the period of time as an indication of the potency of the CD73 antagonist.

(b) measuring the amount of AMP with an AMP-Glo™ Assay in the first and second populations of cells after the period of time; and

(c) comparing the amount of AMP determined in (c) to derive the potency of the CD73 antagonist.

(h) measuring the amount of AMP with an AMP-Glo™ Assay in the cells after the period of time; and

In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises: (a) contacting 4, 5, 6, 7, 8 or more increasing concentrations of (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate (AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) creating a dose-response curve based on the level of AMP at each concentration and for each of (i) the CD73 antagonist and (ii) the reference standard CD73 antagonist, after the period of time, wherein the level of AMP is determined by measuring the amount of AMP with an AMP-Glo™ Assay; and

(b) adding a detergent to the cells after the period of time; and

(c) measuring the amount of AMP with an AMP-Glo™ Assay as an indication of the potency of the CD73 antagonist.

(a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist and AMP, both for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(c) measuring the amount of AMP with an AMP-Glo™ Assay in the first and second populations of cells; and

In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises: (a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73, and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) adding a detergent to the cells after the period of time;

(c) measuring the amount of AMP with an AMP-Glo™ Assay in the cells; and

(i) permeabilizing the cells with a detergent; and

(ii) measuring the amount of AMP in the cells of (i) with an AMP-Glo™ Assay,

(b) adding Triton™ X-100 to the cells after the period of time; and

In some embodiments, a method or assay for assessing the potency of a CD73 antagonist comprises: (a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist and AMP, both for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(c) measuring the amount of AMP with an AMP-G1o™ Assay in the first and second populations of cells; and

(b) adding Triton X-100 to the cells;

(c) measuring the amount of AMP with an AMP-G1o™ Assay in the cells; and

(i) permeabilizing the cells with Triton™ X-100;

(ii) measuring the amount of AMP in the cells of (i) with an AMP-G1o™ Assay, (c) comparing the two dose-response curves to thereby assess the potency of the CD73 antagonist.

(c) measuring the amount of AMP with an AMP-Glo™ Assay in the cells; and (d) comparing the amount of AMP determined in (c) to derive the potency of the CD73 antagonist.

(i) permeabilizing the cells with Triton™ X-100;

(ii) measuring the amount of AMP in the cells of (i) with an AMP-Glo™ Assay,

(a) contacting a first population of cells expressing human CD73 with the CD73 antagonist and AMP and contacting a second population of the same cells with a reference CD73 antagonist and AMP, both for a period of time and under conditions that allow converison of AMP to adenosine in the absence of a CD73 antagonist;

(b) adding Triton™ X-100 to the first and second populations of cells after the period of time and shaking the cells; (c) measuring the amount of AMP with an AMP-Glo™ Assay in the first and second populations of cells; and

(c) measuring the amount of AMP with an AMP-Glo™ Assay in the cells; and

(i) permeabilizing the cells with Triton™ X-100 and shaking the cells;

(ii) measuring the amount of AMP in the cells of (i) with an AMP-Glo™ Assay,

(i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

(c) comparing the two dose-response curves to thereby assess the potency of the CD73 antagonist. Kits

Provided herein are kits that utilize the methods described herein. In some embodiments, the disclosure provides a kit for determining the potency of a CD73 antagonist, comprising (i) cells expressing human CD73, (ii) AMP and (iii) instructions for contacting the CD73 antagonist with the cells and AMP for a period of time and under conditions that allow the AMP to be converted to adenosine in the absence of a CD73 antagonist and subsequently measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist.

In some embodiments, the kit comprises a reference standard antagonist and instructions for preparing a dose-response curve with the reference standard antagonist for comparison to the CD73 antagonist. In some embodiments, the reference standard antagonist is an anti-CD73 antagonist antibody. In some embodiments, the anti-CD73 antagonist antibody is Ab A. In some embodiments, the reference standard antagonist is the same type of antagonist being measured (e.g., reference antagonist is an anti-CD73 antibody if the potency of an anti-CD73 antibody is being determined). In some embodiments, the kit comprises serial dilutions of a reference standard antagonist for generation of a dose-response curve. In some embodiments, the instructions further comprise a method for comparing the dose-response curve generated with the serial dilutions of a reference standard antagonist to the values generated with the CD73 antagonist to determine the potency of the CD73 antagonist.

In some embodiments, the instructions further comprise measuring AMP with a Promega AMP-Glo™ Assay.

EXAMPLES

Example 1: Overview, Materials and Methods and Initial Parameters

A cell based assay was developed to determine the potency of CD73 antagonists, e.g., anti-CD73 antibodies. Overall, cells expressing CD73 were incubated with the CD73 antagonist and AMP, which caused the AMP to be converted to adenosine by unbound or free CD73 (i.e., CD73 that is not bound by the CD73 antagonist), and any remaining AMP was measured by its conversion into ATP, which was used to drive firefly luciferase activity. The resulting luminescent signal (which is proportional to remaining AMP in the system and inversely proportional to the potency of the CD73 antagonist) was measured and the potency of the CD73 antagonist was determined. FIG. 1 provides an overview of the assay. The assay was then modified by the addition of a reagent that permeabilizes the cells after the reaction and prior to measuring the amount of remaining AMP. The reagent that was chosen was Triton X-100, and the assay using Triton X-100 is described below. As shown in FIG. 2, the addition of Triton X-100, allowed the level of remaining AMP to be measured.

Thus, the use of a reagent that permeabilizes the cells, e.g., Triton X-100, after the reaction of the cells with the CD73 antagonist and AMP, was found to be important in this assay.

The assay with Triton X-100 was conducted essentially as follows. A microplate containing SNU-387 cells, which constitutively express human CD73, were treated with increasing concentrations of CD73 antagonist Ab A and, in separate wells, with increasing concentrations of reference standard CD73 antagonist. This was followed by the addition of AMP. Following a period of conversion of AMP to adenosine by CD73, the cells were permeabilized by the addition of the detergent Triton™ X-100. The mixture was then treated with AMP-Glo reagent I, which converted AMP into ADP (adenosine diphosphate) and eliminated any ATP from the cells. Addition of AMP Detection Solution (also referred to as AMP-Glo reagent II) converted the ADP into ATP. The Detection Solution also contained both firefly luciferase and its substrate D-luciferin. The presence of ATP allowed the firefly luciferase to oxidize D-luciferin, producing light, which was measured in a luminometer.

SNU-387 cells (a human hepatocellular carcinoma cell line that endogenously expresses CD73; ATCC Accession NO. CRL-2237) were grown in RPMI 1640 with glutamine (Gibco Catalog No. 11875-093), 10% heat inactivated FBS (Gibco Catalog No. 10082147), and 1% penicillin- streptomycin (Gibco Catalog No. 15140122). CD73 antagonist reference standards, quality controls, and test sample were prepared in diluent Buffer A (RPMI 1640 with 0.01% BSA (Sigma-Aldrich Catalog No. A7979)). AMP (from the AMP-Glo™ Assay kit from Promega (Catalog No. V5012)) and Triton™ X-100 were prepared in diluent Buffer B (0.01% BSA in dPBS with Ca2+ and Mg2+ (Gibco Catalog No. 14040133)).

In the instant Examples, Ab A was used as an exemplary CD73 antagonist. Ab A is an anti-CD73 antagonist antibody that binds specifically to human CD73 and comprises the heavy and light chain variable amino acid sequences set forth as SEQ ID NOs: 135 and 12, respectively, in WO 2016/081748, which is specifically incorporated by reference herein). The full length heavy and light chain amino acid sequences are as follows: Heavy chain:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILYDGSN

KYYPDSVKGRFTI

SRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSSASTKGPS

VFPLAPCSRSTS

ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ

TYTCNVDHKP

SNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

PEVKFNWYVD GVE VHNAKTKPREEQYNST YRVVS VLTVLHQDWLN GKE YKCKV SNKALPS SIEKTIS K AKGQPREPQVYT

LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

TVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1)

Light chain:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPS

RFSGSGSGTD

FTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFY

PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFN

RGEC (SEQ ID NO: 2)

Further description of the assay is provided below, several steps of which were analyzed as described below. While being maintained in a homogenous suspension by frequent mixing by pipetting, the cells were transferred to a microplate comprising various concentrations of the reference standard(s), quality control(s) and/or Ab A, and mixed. AMP (in diluent Buffer B) was added to the cells and incubated for a certain time. Following this incubation, Triton™ X-100 (in Diluent buffer B) was added to the cells. In some experiments, the plate was put on a shaker during cell permeabilization.

The AMP-Glo™ Assay from Promega (Catalog No. V5012) was utilized to measure the amount of AMP remaining after the cell permeabilization step. Specifically, AMP-Glo™ Reagent I and AMP-Glo™ Detection Solution (also referred to herein as AMP-Glo™ Reagent II) were used. Reagent I was first added to the cells and incubated for a period of time, followed by the Detection Solution. After incubation with the Detection Solution, luminescence was measured.

The following calculations were carried out: 1. Response curves for each series of reference standards, Ab A and optional quality control, dilutions were plotted using triplicate absorbance values on the y-axis versus log concentration on the x-axis.

2. Independent 4-parameter logistic curves were fit to the responses using data analysis software to assess curve suitability and parallelism between the reference standard and each of the samples.

3. A constrained 4-parameter logistic curve was fit to the responses using data analysis software to determine potency of each Ab A sample relative to the reference sample. Each sample was independently constrained to the reference standard.

Table 1 provides the initial parameters used in the studies described below. These initial criteria were used to identify important assay parameters and determine preliminary ranges.

Table 1

To determine these initial parameters, the relationship between cell number, AMP concentration and incubation time were explored, and cell numbers and AMP concentrations that would result in about 90% of the AMP being converted into adenosine within a time range that would be robust and easy to control for a quality control (QC) lab. About 90% conversion represents the edge of the linear portion of the AMP conversion range. This represented the starting conditions for the addition of the CD73 antagonist.

We then added a dose response of the CD73 antagonist and optimized the dose response range around the EC₅₀ (prevention of conversion of about 50% of the AMP).

Example 2: Scaling Up to 96 well plates

Although initial experiments with the conditions provided in Table 1 produced acceptable results, showing a reasonable dose response to Ab A, several steps were identified as sources of assay variability. In particular, low volumes of reagents and low numbers of cells contributed to assay variability because small inaccuracies in volumes and cell numbers may result in proportionately larger impacts to the final result. Therefore, to reduce the potential for this influence in the assay, the assay was scaled up to utilize larger volumes in standard 96 well plates.

In the original 1/2 area plate format, preliminary experiments indicated that neither cells/well nor AMP were limiting reagents in the assay. Under conditions of 10,000 cells/well and 10μΜ AMP, the assay produced curves with acceptable accuracy in both half-size, and full size 96 well plates (FIGs. 3A-3B, respectively). Conditions were tested at 40%, 100% and 160% (i.e., about 40%, 100% and 160% of Ab A concentration relative to that of the reference standard) to show assay linearity range. The full area plate conditions were then further optimized.

Example 3: Pre-incubation of Ab A with cells

A critical step in the inhibition of CD73 activity by a CD73 antagonist ( i.e Ab A) is binding of the antibody to its ligand. The dilution scheme for Ab A reference standard started with a concentration of 6μg/mL and further diluted to 2.5 fold to obtain an eight point curve. The final antibody concentration with cells and AMP incubation were at 2 μg/ml to 0.0033μg/mL. To better understand the timeframe under which this inhibition occurs, a pre-incubation step was added. Conditions were tested at 40%, 100%, 130% and 160% concentration to show detection for under potent and hyper potent material. FIG. 4A shows the assay with a preincubation step, and FIG. 4B shows the assay without the preincubation step. Pre-incubation of antibody with cells up to 20 minutes had little effect, indicating that a pre-incubation step is not required. Example 4: Lower asymptote: cells/AMP relationship

An important aspect of the CD73 potency assay is the relationship between CD73 expression and activity on the surface of the SNU-387 cells and the amount of AMP utilized in the assay. If too many cells are present, all of AMP will be converted into adenosine. This will result in an artificial lower asymptote that is representative of complete AMP depletion. Complete AMP depletion over the course of the assay had the potential to impact accuracy by providing an in accurate estimates of EC50. To better understand the relationship between cell number and AMP conversion, the cell numbers required to completely convert 10μΜ ATP into adenosine was assessed. Specifically, various cell numbers were used in the methods described above. Results are shown in FIG. 5. Conditions of 10,000 cells/well and ΙΟμΜ AMP were initially chosen. However, these conditions were shown to result in a hook in the upper asymptote. Therefore, the amount of AMP was subsequently increased to alleviate this hook (see Example 12).

Example 5: Upper asymptote: AMP Glo reagent excess

To achieve an upper asymptote that is representative of remaining AMP in the assay, the AMP-Glo™ reagents must be present in sufficient excess. It was first determined how much AMP could be measured by the amount of AMP-Glo™ reagents present in the assay (60μL/120μL). These experiments were performed in the presence of fully neutralizing amounts of Ab A to accurately reflect conditions of the upper asymptote. As shown in FIG. 6, under conditions of the assay which represent the upper asymptote, the AMP-Glo™ kit behaves linearly between about 1μΜ and about100μM (dashed line). Therefore, a nominal assay concentration of 10μΜ AMP was well within the linear range of the AMP-Glo™ reagents.

It should be noted that at AMP concentrations above 100μΜ, the media was visibly yellow. It is therefore possible that at extremely high AMP concentrations that the assay is adversely affected. However, as 25μΜ is a good concentration to use, this is not a concern for the assay.

Taken together, the nominal condition of 10,000 SNU-387 cells and 25μΜ AMP represents conditions that allows for the generation of reliable lower and upper asymptotes. Example 6: Limits of AMP Glo excess

To ensure consistency of the overall response, the amount of AMP-Glo™ reagents were provided in significant excess. Specifically, various volumes of AMP-Glo™ reagents in the context of high levels of antibody (representing the upper asymptote) and low levels of antibody (representing the lower asymptote). The ratio of AMP-Glo I:AMP-Glo Detection was maintained at 1 :2, according to the manufacturer’s instructions. Although all volumes assessed produced acceptable signal, a signal jump occurred at volumes of 28μL Glo I and was retained through 50μL Glo I. Results are shown in FIG. 7. To ensure retention of signal stability, final volumes of 35μL Glo I and 70μL Detection Solution were chosen for the final conditions.

Example 7: Analyst ease: buffer consistency

An important aspect of development of a QC assay is analyst friendliness. To increase analyst friendliness reagents were diluted in the same media. While the utilization of PBS with 0.01% BSA as a standard diluent buffer improved signal and potency, the amount of BSA could vary between 0.01%-1% with equivalent results. Because PBS is difficult to see in a white walled, white bottom plate, RPMI with 0.01% BSA was substituted for PBS with 0.01% BSA as the diluent buffer for the cells and antibody.

Example 8: Volume scale up

An important aspect of development of a quality control (QC) assay is analyst friendliness. In order to further simplify assay performance, volumes of reagents were increased to support ease of assay set-up and to reduce variability associated with the pipetting of small volumes. The initial and final volumes are indicated in Table 2.

Table 2: Volume comparison

Example 9: Triton X-100 mixing

As described in Example 1, it was determined that the addition of Triton™ X-100 is important for assay performance. Since the ability of Triton™ X-100 to lyse the cells is dependent on both detergent concentration and dispersion, the effects of mixing Triton™ X-100 following addition to the cells was assessed. It was expected that permeabilization should reach a saturating condition where the cell contents reach equilibrium with the surrounding media. The following Triton™ X-100 (Life Technologies) solutions were used: 0.2%, 1% and 2%. Interestingly, while permeabilization appeared to occur whether or not mixing was present, only the shaking condition (5 minutes at ambient room temperature) yielded a saturating condition, where the addition of 25 μL of either 1% or 2% Triton™ X-100 showed similar results (FIG. 8). The mixing condition was therefore deemed an important part of the permeabilization step.

Example 10: Triton X-100 levels

To determine the appropriate range of the detergent to add, various stock concentrations of Triton™ X-100 were prepared (ranging from 0.1% to 2%) and assessed for sufficient permeabilization or potential assay interference. Cells were mixed with the Triton for 5 minutes on a plate shaker. The levels of Triton™ X-100 were tested under assay conditions that represent the upper and lower asymptotes of the assay. Ab A was used at a concentration of 2 μg/mL or 0.0033 μg/mL. Results are shown in FIG. 9. Because a number of Triton™ X-100 levels were acceptable, a 0.5% working stock was settled on, which limited the amount of detergent added to the system, while retaining sufficient permeabilization conditions.

Example 11: Initial assessment of Triton X-100 from Electron Microscopy Sciences

Stock of Triton™ X-100 from Electron Microscopy Sciences was evaluated for equivalent reagent. SNU-387 cells at passage 26 were used to assess the Triton at a final concentration of 0.5%. The results showed all passed system suitability as shown in FIG. 10. Therefore, this Triton stock was used for method validation. During the execution of this Phase Appropriate Validation (PAY), one experiment showed results that were not reflective of the pre- PAY data for curve fit and recovery. Upon investigation, the quality of Triton X-100 utilized was identified as an important factor to method performance, as shown in FIGs. 11A-11B. The experiment was repeated utilizing the same quality of Triton X-100 as the other experiments and the data was found to be representative of pre-PAV performance. Therefore, the origin of the Triton X-100 is an important factor in the assay.

This was confirmed in a separate experiment, conducted as described in the above paragraph of Example 11, and using the high quality Electron Microscopy Sciences Triton X-100 in one assay (FIG. 11C) and a low quality Triton X-100 (FIG. 11D), except that the CD73 antagonist was used at 160% potency (i.e., concentration) relative to the reference standard CD73 antagonist, to be able to differentiate the dose-response curves. As can be seen in FIG. llC, the high quality Triton X-100 produces a dose-response curve with the CD73 antagonist with a distinct shift to the left relative to the dose-response curve of the reference standard, discriminating the over-potent material. However, as can be seen in FIG. 11D, the low quality Triton X-100 is unable to do so for either sample, and the curve quality is noticeably less conforming to a 4 parameter fit and does not meet certain criteria for parallelism/parallel line analysis, including slope.

Example 12: Upper asymptote hook and AMP concentrations

During preliminary assessments of accuracy, an intermittent hook in the upper asymptote was observed (see Example 4, FIG. 5). A number of parameters were assessed for influence on the upper asymptote, including cell number/well, cell passaging scheme, Triton™ X-100 and AMP levels.

When various AMP levels were tested, levels below 10 μΜ (2 μΜ and 5 μΜ) consistently produced an upper asymptote hook, concentrations of 20μΜ and above (20 μΜ, 30 μΜ, and 50 μΜ) did not produce a hook, and 10μΜ AMP variably produced a hook (FIGs. 12A-13F). Interestingly, when viewed together on the same plot, a hook in the upper asymptote is not obvious (FIG. 12A). However, when each curve is plotted individually, the hook becomes evident at lower concentrations of AMP (FIGs. 12B-12F). Therefore, although originally identified as an appropriate standard condition, 10μΜ AMP was on the edge of curve acceptability. Therefore, based on these results, the standard condition was modified to perform the assay at a concentration of 25μΜ AMP. Example 13: Definition of ambient and room temperature

During the cell bank qualification of a specific lot of SNU-387 cells, some of the curves generated from the assays were different from those previously obtained. Among a total of 12 assays, two of these assays failed system suitability. Upon further investigation, it was noticed that room temperature varied significantly. Accordingly, the assays were performed under controlled temperature ranges at 16°C,18°C, 20°C, 22°C, 24°C 25°C, 27°C, 29°C and 37°C. The assays failed system suitability at 16°C, 18 °C, 29 °C and 37 °C (FIGs. 13A-13D) and passed system suitability ranges between 20°C to 27 °C (FIGs. 13E-13I). Based on this, the room temperature for the assay was defined as being between 20°C - 27 °C.

Example 14: Impact of cell passage

To further assess factors that may impact assay suitability, cell passaging was tracked throughout development and assay performance parameters, including signal window and slope. By passage 30, the SNU-387 cells still maintained cell surface expression of CD73, as measured by FACS (FIG. 14A). FIG. 14B shows system suitability in passage 30 cells. Accordingly, a 30 passage limit was implemented. Thus, cells can be used in the assay from passage 3 up to and including passage 30 (p3-p30).

Example 15: Time ranges

The assay has a number of steps with incubation time ranges, including mixing steps after each addition. Each timed step is indicated in Table 3. An experiment bracketing the shortest, nominal and longest incubation times was used to confirm the time ranges indicated for each step in the method. Although the limits of the time ranges were only tested in a single experiment, an assessment of the recovery of samples at the extremes of the linear range of the assay (40% and 160%) under these conditions suggests that the time ranges indicated in the method are justified. Tt should also be noted that the final luminescence signal appears to reach stability by 30 minutes of incubation (Table 3). In addition, the luminescence signal remains stable for at least 60 minutes following a 60 minute incubation with AMP Glo II. Table 3: Incubation and Mixing Time Ranges

Example 16: Exemplary CD73 antagonist potency assay

Based on Examples 1-18 above, an exemplary CD73 antagonist potency assay is set forth below. This assay is written for one CD73 antagonist, but several CD73 antagonists can be tested simultaneously.

1. CD73 expressing cells (e.g., SNU-387 cells) are detached from the tissue culture container (e.g., dish, flask) in which they were grown, and resuspended in Diluent Buffer A at 0.4x10⁶ cells/mL. Taking care to ensure that cells remain in a homogenous suspension by frequent mixing by pipetting, 25 μL of the SNU-387 cells are transferred to each well of the 96-well white wall, white bottom assay plate containing Reference Standard, QC and CD73 antagonist. Note that the cells should have a viability as determined by the cell counter or trypan blue staining to be ≥ 85%. The contents of the wells of the plates are mixed on a plate shaker set at 300 RPM for 60 seconds ± 30 seconds. The final concentration (in μg/mL) of the Reference Standard, Quality Control (QC) and CD73 antagonist in the assay plate(s) containing cells are displayed in Table 4.

Table 4: Final Assay Plate Map for Reference Standard, QC and two CD73 antagonists

2. 25 μL of 75 μΜ AMP (diluted from a 10 mM solution with Diluent Buffer B) is added to each plate row (containing the cells and CD73 antagonist). The final concentration of AMP in each well is 25 μΜ. The contents of the wells of the plates are mixed on a plate shaker set at 300 RPM for 60 seconds ± 30 seconds. The plates are then incubated for 60 minutes ± 10 minutes at ambient temperature (20-27°C) while being protected from the light. Note that the AMP solution may be added up to 20 minutes after the antibody is mixed with the cells.

3. The cells are then lysed by adding 25 μL 0.5% Triton™ X-100 (diluted from a 1% stock solution of Triton™ X-100 in Diluent Buffer B) to each well. The assay plate(s) are shaken on a plate shaker set at 300 rpm for 5 minutes ± 2 minutes at ambient temperature (20-27 °C), while being protected from the light.

4. 35 μL AMP-Glo™ Reagent I is added to the wells of the plates and the assay plate(s) are shaken on a plate shaker set at 300 rpm for 60 seconds ± 30 seconds. The plates are then incubated for 60 minutes ± 10 minutes at ambient temperature (20-27 °C), while being protected from the light.

5. 70 μL AMP-Glo™ Detection Solution (prepared by mixing 80 μL AMP-Glo™ II with 8000 μL Kinase Glo One Solution) is added to the wells, and the assay plate(s) are shaken on a plate shaker set at 300 rpm for 60 seconds ± 30 seconds. The plates are then incubated for 60 minutes ± 15 minutes at ambient temperature (20-27 °C), while being protected from the light. The assay plate(s) are read within two hours of addition of the AMP Detection Solution using a plate reader with luminescent measurement capability.

6. Data analysis is performed as described in USP 1032 and USP 1034, including the following steps: a. The response curves for each series of reference standard, QC and Test Sample dilutions are plotted using triplicate absorbance values on the y-axis versus log concentration on the x-axis. b. Independent 4-parameter logistic curves are fitted to the responses for the reference standard, QC and CD73 antagonists using data analysis software. These fits are used to assess curve suitability and parallelism between the reference standard and each of the samples. c. A constrained 4-parameter logistic curve is fitted to the responses for the reference standard and each of the samples using data analysis software. These fits are used to determine potency of each sample relative to the reference standard. Each sample must be constrained to the reference standard independently. d. The 4-parameter logistic curve fit is defined by the equation below:

Equation 1:

Where:

A = upper asymptote B = slope factor C = inflection point D = lower asymptote x = concentration of the reference standard/QC/samplc(s) y = Response (RLU)

Claims

1. A method for assessing the potency of a CD73 antagonist, comprising the steps of:

2. A method for quantifying the ability of a CD73 antagonist to prevent enzymatic conversion of AMP to adenosine by human CD73 expressed on a cell, comprising the steps of:

3. An in vitro assay for assessing the activity of a CD73 antagonist comprising the steps of:

4. A method for determining whether a manufactured CD73 antagonist fulfills a predefined criterion, e.g., having a similar potency to that of a reference standard CD73 antagonist, comprising the steps of:

(b) measuring the amount of AMP after the period of time.

5. The method of any one of claims 1-4, further comprising the step of permeabilizing the cells prior to measuring the amount of AMP.

6. The method of claim 5, wherein the step of permeabilizing the cells comprises contacting the cells with a cell permeabiliztion agent, such as a detergent.

7. The method of claim 6, wherein the detergent is Triton™ X-100.

8. The method of claim 6, wherein the detergent is 0.5% to 5% Triton™ X-100.

9. The method of claim 6, wherein the detergent is 0.5% Triton™ X-100.

10. The method of any one of claims 7-9, wherein Triton™ X-100 is 95%-100% pure.

11. The method of any one of claims 6-10, wherein the step of permeabilizing the cells further comprises shaking the cells after the addition of the cell permeabilization agent.

12. The method of any one of claims 1-11, wherein the step of measuring the amount of AMP is determined by (i) adding a first reagent comprising a first enzyme capable of converting the AMP to adenosine diphosphate (ADP); (ii) adding a second reagent comprising a second enzyme capable of converting the ADP to adenosine triphosphate (ATP); and (iii) determining the amount of ATP.

13. The method of claim 11, wherein the first and second reagents are added simultaneously or consecutively.

14. The method of claim 11, wherein the second reagent is added after AMP is converted to

ADP.

15. The method of any one of claims 12-14, wherein the first enzyme is polyphosphate: AMP phosphotransferase (PAP) and the second enzyme is adenylate kinase (AK).

16. The method of any one of claims 12-15, wherein the first and second reagents are Reagent I and AMP Detection Solution, respectively, from an Amp-Glo™ assay.

17. The method of any one of claims 12-16, wherein the ratio of first reagent: second reagent is 1:2.

18. The method of any one of claims 12-17, wherein the step of determining the amount of

ATP comprises adding luciferase and luciferin.

19. The method of any one of claims 1-18, wherein the cells are human cells.

20. The method of any one of claims 1-19, wherein the cells naturally express human CD73 on the cell surface or the cells have been engineered to express human CD73 on the cell surface.

21. The method of any one of claims 1-18, wherein the cells are SNU-387 cells (ATCC

Accession No. CRL-2237) or Calu-6 cells (ATCC Accession No. HTB-56).

22. The method of any one of claims 1-21, wherein about 10,000 cells expressing human

CD73 are used per well of a 96 well plate.

23. The method of any one of claims 1-22, wherein the cells are passaged no more than 30 times.

24. The method of any one of claims 1-23, wherein AMP has a concentration of about 10 μΜ to about 100 μΜ.

25. The method of claim 24, wherein AMP has a concentration of about 15 μΜ to about 35 μΜ.

26. The method of claim 25, wherein AMP has a concentration of about 25μΜ.

27. The method of any one of claims 1-26, wherein the method is performed at a temperature of 20°-27°C.

28. The method of any one of claims 1-27, comprising generating a dose-response curve with the CD73 antagonist and a dose-response curve with a reference standard CD73 antagonist having a known potency, and determining the potency of the CD73 antagonist by comparing the dose- response curves.

29. The method of claim 28, wherein the reference standard CD73 antagonist is an anti-CD73 antagonist antibody which binds to human CD73.

30. The method of claim 29, wherein the reference standard CD73 antagonist is Ab A.

31. A method for assessing the potency of a CD73 antagonist (or “test CD73 antagonist”), comprising the steps of:

(a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, with cells expressing human CD73 and adenosine monophosphate

(AMP) for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist;

(b) measuring the amount of AMP after the period of time by:

(i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent I;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

32. A method for assessing the potency of a CD73 antagonist, comprising the steps of:

(a) contacting (i) the CD73 antagonist and (ii) in a separate vial or well, a reference standard CD73 antagonist, about 10,000 cells expressing human CD73, and 25 μΜ adenosine monophosphate (AMP) for about 60 minutes at 20°-27°C;

(b) measuring the amount of AMP after (a) by:

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

33. A method for assessing the potency of a CD73 antagonist (or “test CD73 antagonist”), comprising the steps of:

(i) permeabilizing the cells with 0.5% Triton X-100;

(ii) contacting the cells of (i) with AMP-Glo™ Reagent T;

(iii) contacting the cells of (ii) with AMP-Glo™ Detection Solution; and

(iv) exposing the cell of (iii) to a luminescent measuring device;

34. A method for assessing the potency of a CD73 antagonist, comprising the steps of:

20°-27°C;

(iv) exposing the cells of (iii) to a luminescent measuring device;

35. The method of claim 33 or 34, wherein step (a) comprises contacting 8 concentrations ranging from 2 μg/ml to 0.0033μg/mL of the CD73 antagonist and the reference standard CD73 antagonist with the cells expressing human CD73 and AMP.

36. A kit for determining the potency of a CD73 antagonist, comprising cells expressing human CD73, AMP and instructions for contacting the CD73 antagonist with the cells and AMP for a period of time and under conditions that allow conversion of AMP to adenosine in the absence of a CD73 antagonist and subsequently measuring the amount of AMP after the period of time as an indication of the potency of the CD73 antagonist.

37. The kit of claim 36, further comprising a reference standard CD73 antagonist and instructions for preparing dose-response curves with the CD73 antagonist and the reference standard CD73 antagonist.

38. The kit of claim 37, wherein the reference standard antagonist is an anti-CD73 antagonist antibody.

39. The kit of claim 38, wherein the anti-CD73 antagonist antibody is Ab A.