Download Flow Cytometry - From Discovery to Clinical Analysis | Charles River

Issue 224 | June 2014
Flow Cytometry – From Discovery to Clinical Analysis
Flow cytometry is a laser-based technology that analyzes multiple characteristics of a single particle (usually cells). It allows
multiparametric analysis of thousands of particles per second and helps to adequately identify or functionally characterize
complex cell populations of interest.
Flow cytometry is a versatile technology for developing drug-specific assays. It is often used in basic research, discovery,
preclinical and clinical trials. With the increasing proportion of biologics in the pipeline, flow cytometry has proven itself to be an
indispensable tool in many cases to assess safety, receptor occupancy or pharmacodynamics.
In discovery, flow cytometry technology can be used to screen drug candidates for changes in immunophenotype or cell function
using ex vivo or in vivo designs. Plate-format assays requiring only small amounts of samples and/or drugs can de developed,
using multicolor fluorescence and high-throughput acquisition.
During the preclinical phase, flow cytometry has routinely been used for assessing the immunotoxicology of a candidate drug
by evaluating the immunophenotype of various cell populations in whole blood, tissue or other matrices. In addition, receptor
occupancy (RO) is commonly included in preclinical programs to evaluate the binding of the drug to target cells. Flow cytometry
can also be used to assess pharmacodynamic (PD) markers of interest for further elucidation of on- or off-target effects.
In a clinical setting, flow cytometry can be used for safety, RO and PD assessments. Flow cytometry has also been used to
investigate the mechanisms of action of toxicity.
DISCOVERY
• Functional assays
(efficacy assays)
PRECLINICAL
CLINICAL
•Safety
(immunophenotyping,
functional assays, nAb assays)
• Receptor occupancy
•Pharmacodynamics
(functional assays)
•Safety
(immunophenotyping,
functional assays)
• Receptor occupancy
•Pharmacodynamics
(functional assays)
The expert scientific staff at Charles River can provide services for flow cytometry method transfer, method development,
method validation, and high-quality sample analysis and interpretation in accordance with Good Laboratory Practices (GLP)
requirements and applicable clinical GLP requirements. Our immunology groups provide services to support clients’ need to
evaluate the potential effect of their compound on various cell populations and/or functions. Our experience includes evaluation
of various types of test articles, including, but not limited to, monoclonal antibodies, antibody drug conjugates, small molecules,
proteins and peptides. Furthermore, Charles River can develop novel flow cytometric assays as needed, if appropriate reagents
are available, that can be carried through from discovery to the clinical phase. Fluorochrome conjugation services are also
available if the reagent is not available commercially.
Our Preclinical Services group has expertise in flow cytometry at all 3 levels of drug development, from discovery screening
to clinical analysis. In this Researcher, we present several types of assays developed by Charles River and offered as general
assays or developed specifically for a compound.
download previous Researcher issues on The SourceSM, please visit www.criver.com/thesource
Immunophenotyping
Immunophenotyping is the analysis of heterogeneous cell
populations to identify the presence and proportions of
various populations of interest. Antibodies are used to identify
cells by detecting specific markers expressed by these cells
(cell surface markers or intracellular markers). Changes in
these populations, as an effect of the drug, are observed as
depletion, activation, expansion or migration to various organs.
Immunophenotyping can be performed in various tissues
(e.g., thymus, spleen, lymph node, bone marrow), in peripheral
blood, and broncheoalveolar fluid (BALF).
Immunophenotyping of T, B and NK cells is a common
endpoint added to preclinical studies in order to assess the
potential immunotoxicity effect of a drug. While changes in
cell phenotypes are useful in some settings to characterize
the immunotoxicity of different compounds, phenotypic
analysis alone is often not a sensitive indicator of low-dose
immunotoxicity for many agents that alter immune function.
Substances that exert selective toxicity on lymphoid and
myeloid cells may be discovered through immunophenotypic
analysis. However, most agents produce immunotoxicity at
doses much lower than those required to produce cytotoxicity
or interfere with primary lymphoid organ differentiation.
Some of the most potent immunosuppressive chemicals
that have been tested, such as cyclosporine A, do not alter
the number of cells/immunophenotype at doses that are
immunosuppressive, even though the immune cell function is
altered. In these cases, immunophenotyping can be assessed
in conjunction with functional parameters such as a cytoxic
T-cell assay or a natural-killer cell activity assay to identify the
immunotoxic effects.
Immunophenotyping may also, in some instances, be used
as a pharmacodynamic endpoint. Indeed, some drugs, such
as rituximab, are designed to specifically deplete lymphocyte
populations. In such cases, immunophenotyping is a sensitive
endpoint to monitor the efficacy of the drug.
The list of cell surface markers or intracellular markers is
growing fast and is practically limitless, given that any antibody
can be labeled with a variety of fluorochromes for potential
flow cytometry uses whenever pre-labeled antibodies are not
already available. Although the availability of antibodies is
vast, it should be taken into account when designing studies
that availability of antibodies reactive in species like canine,
swine and/or rabbit will be limited compared to antibodies for
humans, NHPs or rodents.
Identification of small populations
Flow cytometry is often a more sensitive platform for the
detection of small cell populations. For example, regulatory
T cells represent only 1-2% of total lymphocytes in peripheral
blood, but these cells serve a vital immunosuppressive
function. Monitoring the expression of regulatory T-cell markers
CD25 and FoxP3 during administration of a drug is important
for determination of immunotoxicity (e.g., immunomodulation,
oncology drugs). Bone marrow (BM)-derived CD34+
hematopoietic stem cells are another example of a small
population that can be identified by flow cytometry at
concentrations as low as < 5 cells per µL. Many other cell
populations, such as iNKT and dendritic cells, have also been
analyzed by flow cytometry, generally to demonstrate the
efficacy of a specific drug.
Tetramer technology
The tetramer technology allows the identification of T cells that
respond to a particular antigen. MHC tetramers are formed by
first refolding MHCs in the presence of high concentrations
of the desired antigenic peptide, followed by biotinylation of
the carboxy-terminus of one chain of the MHC molecule. This
MHC/peptide complex can then be bound to streptavidin and
exposed to T cells. The use of fluorophore-labeled streptavidin
for tetramer formation allows for efficient detection by flow
cytometry. The use of the tetramer technology facilitates
accurate discrimination of rare, specific T cells (less than 1%
of the population). Additional markers can be used to further
characterize the cell population binding the specific peptide
tested. As the peptide of interest is drug-dependent, tetramer
assays need to be developed for each specific project.
Micronucleus
Flow cytometry can also be used to improve the throughput
of traditional but less effective methodology. One example is
the manual micronucleus method, which is used as a genetic
toxicology parameter. The conversion of this method to flow
cytometry readout results in a more efficient assay in which a
larger number of red blood cells is used for scoring (100 vs.
> 10,000 cells).
The Litron PigA assay measures genetic toxicity by detecting
mutations in the PigA gene, which is required for the synthesis
of GPI anchors for cell surface proteins. The PigA gene is
located on the X chromosome; therefore, male (XY) individuals,
who have only a single copy of the gene, and females (XX),
who also have only one functional copy of the gene due to
X-chromosome inactivation, both lose cell surface expression
GPI-anchored proteins as a result of a single inactivating
mutation of the gene. Mutations in PigA are neutral and do not
convey any survival advantage or disadvantage; therefore the
incidence of PigA mutations should accurately reflect mutation
rates.
The PigA assay uses flow cytometry to measure the expression
of CD59 on erythrocytes and reticulocytes; a GPI-anchordeficient phenotype indicates PigA mutation in bone marrow
precursor cells. The analysis requires small volumes of blood
from treated animals and does not require sacrifice; the test is
therefore suitable as an add-on to existing long-term studies.
Functional Assays
Functional flow cytometry endpoints can be simple or complex
assays. In general, these assays require a cell function which
can be detected by antibodies towards a protein or marker of
the functional pathway of interest, a dye which can discriminate
a cell’s status or activity, or by an enzyme-substrate reaction.
These assays can be carried out to determine in vivo, ex vivo
(with an in vitro stimulation), or in vitro functions of the target
cell of interest. Below are examples of functional assays using
flow cytometry.
Oxidative burst/phagocytosis
Phagocytosis by neutrophils and monocytes constitutes an
essential arm of innate immunity against bacterial or fungal
infections. The phagocytic process can be separated into
several major stages: chemotaxis (migration of phagocytes to
inflammatory sites), attachment of particles to the cell surface
of phagocytes, ingestion (phagocytosis) and intracellular
killing by oxygen-dependent (oxidative burst) and oxygenindependent mechanisms. The phagocytosis assay consists
of measuring the amount of fluorescent bacteria ingested by
phagocytes to quantitatively determine the ability of leukocytes
to function in the presence of a drug. After phagocytosis,
granulocytes and monocytes produce reactive oxygen
metabolites which destroy bacteria inside the phagosome.
The oxidative burst assay measures the formation of the
reactive oxidants by the enzymatic oxidation of a fluorogenic
substrate, DHR 123. These assays can be used both as a
safety or an efficacy endpoint.
Figure 1. Granulocyte function by flow cytometry
Oxidave burst
Baseline
Normal sample (E.Coli)
Inhibited sample (E.Coli + Cyto D)
Posive control (PMA)
Phagocytosis
Baseline
Normal sample (E. coli)
Inhibited sample (E. coli + Cyto D)
Indicators of cell death and injury
There are many fluorescent probes that can be used in flow
cytometry as indicators of cell injury and stress. The selection
of these markers should be based on the utility of the intended
data. Markers available include those for membrane integrity,
cell viability, death pathways, oxidative stress as well as other
stress markers. Indication of cell injury or death can be used
for safety evaluation in vivo as well as in vitro assays to assess
the impact of a drug on target cells.
Basophil activation
Both true allergic and pseudo-allergic reactions can result in
anaphylactoid-type symptoms because common mediators
are released (e.g., histamine leukotrienes, etc.). It is often not
possible to implicate IgE in a direct role for many adverse
reactions. The basophil activation test performed by flow
cytometry can give an indication of a Type I-like hypersensitivity
reaction. In this type of assay, basophils are identified as
CCR3+, and upon activation, CD63, a marker present within
the basophil granules, becomes externalized and presented
on the surface of the cells. Activated basophils can then be
monitored through the level of CD63 expressed at the surface.
Figure 2. Basophil degranulation by flow cytometry
Negave control
Figure 3. Platelet activation by flow cytometry
Resng platelets
Acvated platelets
SSC
SSC
Resng
platelets
CD61
CD61
Pac 1 +
Act platelets
PAC-1
PAC1
Intracellular protein expression
A variety of antibodies are available to study intracellular
protein expression in different cell class subtypes.
When combined with surface marker expression in lymphoid
cells, these assays become extremely powerful for assessing
the effects of drugs on defined cell populations. Examples of
assays that can be performed in lymphoid subsets include
intracellular cytokine expression and various cell signaling
expression ranging from general phosphotyrosine to specific
phosphoproteins. Key phosphorylation pathways, such as
those involving the molecules p38, ERK, MEK, PKCs, IP3,
ZAP70, etc., can be identified for specific cell subsets
(lymphocytes, phagocytes, basophils and eosinophils) and
can be included in standard flow cytometry assays to identify
specific phosphorylation cascades modulated by various
compounds. Currently, the most widely-used phospho-specific
antibodies for T-cell functions are antibodies targeting MAP
kinase phosphorylation and STAT protein phosphorylation.
fMLP
Platelet activation
Flow cytometry is not limited to the evaluation of immune cell
functions. Methods for functional assessment of other types
of cells are available. For example, flow cytometric analysis of
platelets is commonly used to study many aspects of platelet
biology and function (in vivo and ex vivo). Isolated platelets or
anticoagulated whole blood can be incubated with a variety of
reagents that bind specifically to individual platelet proteins,
granules and lipid membranes to monitor the activation state
of platelets. Platelet activation triggers significant remodeling
of the platelet cell surface. Among the markers used to
monitor platelet activation, we find P selectin (CD62P), the
GPIIb/IIIa complex (PAC-1), fibrinogen, and the measurement
of platelet-leukocyte aggregates. Charles River is uniquely
capable of conducting studies using platelet activation assays
as endpoints, as these assays require immediate processing
and special handling in the laboratory, which is located on the
same premises as the vivarium.
PAC1
Cell activation
Expression of many adhesion molecules and growth
receptors is altered on leukocytes during cell activation and
differentiation. Many of these molecules are lineage-associated
and can be used to identify subsets as well as altered
expression following exposure to drugs. Several examples
of adhesion molecules that are up- or down-regulated in
response to immune activation or toxicants exist, the most
common being CD25 (IL-2R), CD62L (L-selectin) and CD69
(C-type lectin). Other markers of cell activation and proliferation
that are being increasingly utilized include various cytokine and
chemokine receptors and proliferating cell antigens such as
Ki67 protein.
An-FcεRI Ab
Resng
platelets
CD62P
CD62P
CD62P+
Act platelets
CD62P
Receptor Occupancy
As many monoclonal antibody drug therapeutics bind to
specific cell surface receptors, it is often useful to quantify
the receptor occupancy of a specific cell subpopulation by
the drug. Typical approaches involve the use of competitive
(binding is blocked by the investigational drug) and noncompetitive (binds to a different epitope of the antigen
targeted by the investigational drug) fluorochrome-conjugated
monoclonal antibodies (see Figure 1). The end result is a
customized assay for a particular biological drug, which may
yield useful information relevant to dosage, safety and efficacy.
Figure 4. Receptor occupancy assay design by flow cytometry
Unlabeled rituximab or
rituximab-MMAE/TA
FITC-labeled rituximab or
rituximab-MMAE/TA
Platelet-bound immunoglobulins
Immune-mediated platelet destruction is a common cause
of thrombocytopenia. Immune-mediated platelet destruction
occurs when circulating platelets coated with antibody,
antigen-antibody complexes or complement are phagocytozed
by macrophages. Some drugs have the capability to induce
the production of antibodies binding to various structural
platelet antigens, resulting in accelerated platelet destruction.
The quantitation of platelet-associated immunoglobulin by
flow cytometry is helpful to confirm a diagnosis of immunemediated thrombocytopenia; it can also determine the
subclass of immunoglobulins (IgA, IgM or IgG) binding the
platelets.
Figure 6. Detection of platelet-bound antibodies by flow cytometry
Pre-Dose
Post-Dose
Negative control
Human Anti-IgA
Human Anti-IgM
Human Anti-IgG
CD20
CD19
Anti-CD19
Immunogenicity
Immunogenicity refers to the ability of a drug to induce
an immune response. The concerns associated with an
immunogenic response are that anti-drug antibodies (ADA)
could potentially:
• increase or decrease the clearance of a drug,
• form aggregates with a drug and induce toxicity,
• alter the biological activity of a drug through the
formation of neutralizing antibodies (nAb),
• cross react with endogenous proteins, or
• induce drug allergenicity through the formation of
anti-IgE ADA.
A measurement of ADA is routinely done by ELISA or ECL.
However, when the neutralizing potential of these antibodies
needs to be determined, a more complex bioassay needs to
be developed. Some of these assays utilize a flow cytometrybased platform.
Figure 5. Neutralizing antibodies by flow cytometry
Neutralizing
Mean
anbody amount Fluorescence
(µg/mL)
Intensity
0
791
3.13
397
6.25
144
12.5
19
25.0
11
50.0
8
Black line = unstained cells
--
nAb amount
MFI
++
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Advantages of outsourcing flow cytometry assays:
- Highly experienced scientists willing to work as a
member of your team for the development of novel
assays
- Significant degree of standardization for custom
cell-based assays implemented globally
- Identical flow cytometry platforms available to perform all
assays
- Validated SOP-driven methods
- Minimized variation in the final data set,
as interinstrument and interanalyst variability is assessed
during validation; this increases the standardization of
results generated
- Time savings
- Trained personnel provide access to resources not
available internally
- Internal resources freed up for other purposes
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