Flow cytometry and receptor occupancy in immune-oncology

Figures & data

Figure 1. Three types of measurements for flow-based ROA. Free Receptor Assay: can be measured using a fluorescent conjugated competitor of the therapeutic or a directly labeled therapeutic to detect unbound. Bound Receptor Assay: can be measured using a specific anti-therapeutic antibody. Total Receptor Assay: can be measured using a non-competing antibody or saturating amount of unlabeled therapeutic.

Figure 2. A. Labeled therapeutic spiked into the surrogate sample at saturating concentration. The histogram on the right side shows a saturated signal as indication of the therapeutic binding to all the targets on the cell surface. B. One or more concentration of therapeutic B spiked into the sample surrogate with saturating concentration of labeled therapeutic. The histogram on the right side shows a decrease in signal of the labeled therapeutic as indication of therapeutic B interference of target binding. C. Fluorescent-labeled therapeutic is spiked into the surrogate sample with pHrodo labeled therapeutic. If the target is internalized pHrodo is activated and a green-fluorescent signal will be detected. The fluorescent-labeled therapeutic will bind the target on the surface of the cells.

Table 1. Flow cytometry panel design and RO calculation

Flow cytometry Panel design	RO calculation
1. Labeled anti-ID 2. FMO control/IC control	%RO = bound/total X100 (% or MFI or MESF)
1. Labeled non competing antibody 2. FMO control/IC control	%RO = 1 -(Bound/Total)x100 (% or MFI or MESF)
1. IPT panel 2. FMO control/ IC control	%RO = 1 -(post-treatment/pre-tratment)x100 (% or MFI or MESF)
1. IPT panel 2. FMO control/ IC control	%RO = {(total- 100)- (free-100)/(total-100)}x100 (% or MFI or MESF)

Table 2. ROA targets, development stage, and purposes

	Assay format	Reagent	Development stage	Purpose	Study
CD28	Bound	aPEG mAb	Preclinical	PD biomarker	[18]
PD-1	Bound	ahuIgG4	Phase 1	PD biomarker	[19]
CD20	Free	Competing mAb	Preclinical	PD biomarker	[11]
CD22	Free	Competing mAb	Phase 1	PD biomarker	[20]
CD28	Free	Labeled Drug	Preclinical	PD biomarker	[21]
CD4	Free	Labeled Drug	Phase 1	PD biomarker	[22]
CD40	Free	Not determined; Labeled drug	Phase 1 and Preclinical	PD biomarker	[10,23–25]
IGF-1 R	Free	Competing mAb	Phase 1	PD biomarker	[26]
IL-7Rα	Total and Free	Labeled Drug		PD biomarker	[27,28]
KIR	Free	Labeled Drug	Phase 1	PD biomarker	[29]
LFA1	Free	Competing mAb	Preclinical	PD biomarker	[12]
PD-1	Bound	anti-idiotype for RG7769	Phase 1	PD biomarker	[30]
JS001 (anti-PD-1)	Bound	ahuIgG4	Phase 1	PD biomarker	[28]
SAR3419 (antibody drug conjugate targeting CD19)	Total	muαB4	Phase 1	PK/PD	[31]
AVE9633 (antibody drug conjugate targeting CD19)	Total	Labeled Drug	Phase 1	PK/PD	[32]

Table 3. ROA design scheme and considerations

ROA design scheme	Considerations
Biology of the therapeutic	Induce receptor internalization and/or shedding
Mechanism of action of the therapeutic	downstream signaling pathways
Availability of reagents	competitive antibody, noncompetitive antibody, anti-idiotype (preferable), labeled drug, secondary antibody for drug detection (high background)
Harmonization of the immunophenotyping panel	assess compatibility between fluorochrome and antibodies in the Immunophenotyping panel (reactivity check)
Biological variability	perform titration curves of the reagents using at least 3 donors
Abundance or rarity of target expression	the saturating concentrations may vary according to the level of expression of the target. Suggested controls: cell lines for low, medium, high expression of the target, surrogate samples and QC controls.
Frequency and stability of the target expressing cell population/s	circulating or noncirculating targets
Specimen type and conditions	Impact of fresh versus frozen, sample tube collection, anti-coagulant, temperature shipping conditions
Reproducibility of results	Assessment of changes in specimens collected at different times over the course of the therapy and standardization of the instrumentation

Table 4. Validation parameters, samples, and acceptance criteria

Validation Parameters	Samples	Acceptance criteria
Intra-Assay Precision	1 Donors	%CV ≤ 25%/35%
	1 Analytical Run
	6 Replicates
Inter-Assay Precision	3 Donors	%CV ≤ 25%/35%
	3 Analytical Runs
	3 Replicates
Sample Stability (unstimulated)	3 Donors	Change from baseline ≤ 25%/35%
	3 Analytical Runs (0 hr, 24 hr, 48 hr)
	3 Replicates
Sample Stability (stimulated)	3 Donors	Change from baseline ≤ 25%/35%
	3 Analytical Runs (0 hr, 24 hr, 48 hr)
	3 Replicates
Fixed Sample Stability 4°C	1 Donor	Change from baseline ≤ 25%/35%
	3 Analytical Runs (0 hr, 24 hr, 48 hr, 72 hr post-fix)
	3 Replicates

Suchard SJ, Davis PM, Kansal S, et al. A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety. J Immunol. 2013;191(9):4599–4610.

 PubMed Web of Science ®Google Scholar

Brahmer JR, Drake CG, Wollner I, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol. 2010;28(19):3167–3175.

 PubMed Web of Science ®Google Scholar

Mao C-P, Brovarney MR, Dabbagh K, et al. Subcutaneous versus intravenous administration of rituximab: pharmacokinetics, CD20 target coverage and B-cell depletion in cynomolgus monkeys. PLoS One. 2013;8(11):e80533.

 PubMed Web of Science ®Google Scholar

Advani A, Coiffier B, Czuczman MS, et al. Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin’s lymphoma: results of a phase I study. J Clin Oncol. 2010;28(12):2085–2093.

 PubMed Web of Science ®Google Scholar

Waibler Z, Sender LY, Merten C, et al. Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies. PLoS One. 2008;3(3):e1708.

 PubMed Web of Science ®Google Scholar

Zheng Y, Scheerens H, Davis JC, et al. Translational pharmacokinetics and pharmacodynamics of an FcRn-variant anti-CD4 monoclonal antibody from preclinical model to phase I study. Clin Pharmacol Ther. 2011;89(2):283–290.

 PubMed Web of Science ®Google Scholar

Ma A, Dun H, Song L, et al. Pharmacokinetics and pharmacodynamics of ASKP1240, a fully human anti-CD40 antibody, in normal and renal transplanted Cynomolgus monkeys. Transplantation. 2014;97(4):397–404.

 PubMed Web of Science ®Google Scholar

Tolcher AW, Sarantopoulos J, Patnaik A, et al. Phase I, pharmacokinetic, and pharmacodynamic study of AMG 479, a fully human monoclonal antibody to insulin-like growth factor receptor 1. J Clin Oncol. 2009;27(34):5800–5807.

 PubMed Web of Science ®Google Scholar

Kern B, Li W, Bono C, et al. Receptor occupancy and blocking of STAT5 signaling by an anti-IL-7 receptor α antibody in cynomolgus monkeys. Cytometry B Clin Cytom. 2016;90(2):191–198.

 PubMed Web of Science ®Google Scholar

Fu J, Wang F, Dong L-H, et al. Receptor occupancy measurement of anti-PD-1 antibody drugs in support of clinical trials. Bioanalysis. 2019;11(14):1347–1358.

 PubMed Web of Science ®Google Scholar

Vey N, Bourhis J-H, Boissel N, et al. A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission. Blood. 2012;120(22):4317–4323.

 PubMed Web of Science ®Google Scholar

Woska JR Jr., Last-Barney K, Rothlein R, et al. Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay. J Immunol Methods. 2003;277(1–2):101–115.

 PubMed Web of Science ®Google Scholar

Junker F, Gulati P, Wessels U, et al. A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1. Cytometry A. 2021. DOI:https://doi.org/10.1002/cyto.a.24334.

 Google Scholar

Ribrag V, Dupuis J, Tilly H, et al. A dose-escalation study of SAR3419, an anti-CD19 antibody maytansinoid conjugate, administered by intravenous infusion once weekly in patients with relapsed/refractory B-cell non-Hodgkin lymphoma. Clin Cancer Res. 2014;20(1):213–220.

 PubMed Web of Science ®Google Scholar

Lapusan S, Vidriales MB, Thomas X, et al. Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia. Invest New Drugs. 2012;30(3):1121–1131.

 PubMed Web of Science ®Google Scholar