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EMEA 2006 Reproduction and/or distribution of this document is authorised for non commercial purposes only provided the EMEA is acknowledged
May 2006
CHMP/167235/2004
ICH Topic S 8
Immunotoxicity Studies for Human Pharmaceuticals
Step 5
NOTE FOR GUIDANCE ON IMMUNOTOXICITY STUDIES FOR HUMAN
PHARMACEUTICALS
(CHMP/167235/2004)
TRANSMISSION TO CHMP December 2004
TRANSMISSION TO INTERESTED PARTIES December 2004
DEADLINE FOR COMMENTS April 2005
FINAL APPOVAL BY CHMP October 2005
DATE FOR COMING INTO OPERATION May 2006
© EMEA 2006 2
IMMUNOTOXICITY STUDIES FOR HUMAN PHARMACEUTICALS
Table of Content
1. INTRODUCTION............................................................................................................................... 3
1.1 OBJECTIVES OF THE GUIDELINE.................................................................................................... 3
1.2 BACKGROUND .............................................................................................................................. 3
1.3 SCOPE OF THE GUIDELINE ............................................................................................................ 4
1.4 OVERVIEW .................................................................................................................................... 4
2. GUIDELINE......................................................................................................................................... 4
2.1 FACTORS TO CONSIDER IN THE EVALUATION OF POTENTIAL IMMUNOTOXICITY ............................ 4
2.1.1 Standard Toxicity Studies..................................................................................................... 5
2.1.2 Pharmacological Properties ................................................................................................ 5
2.1.3 Intended Patient Population................................................................................................. 5
2.1.4 Structural Similarity............................................................................................................. 6
2.1.5 Disposition of the Drug ........................................................................................................ 6
2.1.6 Signs Observed in Clinical Trials or Clinical Use............................................................... 6
2.2 WEIGHT OF EVIDENCE REVIEW .......................................................................................................... 6
3. SELECTION AND DESIGN OF ADDITIONAL IMMUNOTOXICITY STUDIES ..................... 6
3.1 OBJECTIVES....................................................................................................................................... 6
3.2 SELECTION OF ASSAYS...................................................................................................................... 6
3.3 STUDY DESIGN.................................................................................................................................. 7
3.4 EVALUATION OF ADDITIONAL IMMUNOTOXICITY STUDIES AND NEED FOR FURTHER STUDIES ..... 7
4. TIMING OF IMMUNOTOXICITY TESTING IN RELATION TO CLINICAL STUDIES....... 7
REFERENCES........................................................................................................................................... 7
APPENDIX: METHODS TO EVALUATE IMMUNOTOXICITY.................................................... 9
© EMEA 2006 3
IMMUNOTOXICITY STUDIES FOR HUMAN PHARMACEUTICALS
1. INTRODUCTION
1.1 Objectives of the guideline
The objectives of this guideline are to provide (1) recommendations on nonclinical testing
approaches to identify compounds which have the potential to be immunotoxic, and (2)
guidance on a weight-of-evidence decision making approach for immunotoxicity testing.
Immunotoxicity is, for the purpose of this guideline, defined as unintended
immunosuppression or enhancement. Drug-induced hypersensitivity and autoimmunity are
excluded.
1.2 Background
Evaluation of potential adverse effects of human pharmaceuticals on the immune system
should be incorporated into standard drug development. Toxicity to the immune system
encompasses a variety of adverse effects. These include suppression or enhancement of the
immune response. Suppression of the immune response can lead to decreased host resistance
to infectious agents or tumor cells. Enhancing the immune response can exaggerate
autoimmune diseases or hypersensitivity. Drug or drug-protein adducts might also be
recognized as foreign and stimulate an anti-drug response. Subsequent exposures to the drug
can lead to hypersensitivity (allergic) reactions. Much of the science and method development
and validation efforts in the past have been focused on evaluating drug development
candidates for their potential for either immunosuppression or contact sensitization. No
standard approaches for human pharmaceuticals are currently available for testing for
respiratory or systemic allergenicity (antigenicity) or drug-specific autoimmunity; testing for
these endpoints is not currently required in any region. There are no regional differences in
testing approaches of skin sensitization.
Immunosuppression or enhancement can be associated with two distinct groups:
1) Drugs intended to modulate immune function for therapeutic purposes (e.g. to prevent
organ transplant rejection) where adverse immunosuppression can be considered
exaggerated pharmacodynamics.
2) Drugs not intended to affect immune function but cause immunotoxicity due, for
instance, to necrosis or apoptosis of immune cells or interaction with cellular receptors
shared by both target tissues and non-target immune system cells.
Anti-proliferative agents used to treat cancer are an example of drugs that produce unintended
immunosuppression. In such instances, adverse findings in nonclinical studies are predictive
of human immunotoxicity in a rather straightforward manner. That is, specific assays to
determine immunotoxicity are probably not valuable in drug risk assessment since the target
tissues are usually rapidly dividing cell types, such as bone marrow-derived immune system
progenitor cells. Hence, the adverse effects on immune function can be predicted based on
pharmacologic activity and can usually be reliably evaluated in non-clinical studies. For other
types of compounds not intended to suppress the immune response, distinction between
exaggerated pharmacodynamics and non-target effects can be less obvious. As an example,
some anti-inflammatory compounds have an effect on certain innate immune functions but do
not necessarily affect the adaptive immune response.
© EMEA 2006 4
1.3 Scope of the Guideline
This guideline is focused on providing recommendations on nonclinical testing for
immunotoxicity induced by human pharmaceuticals. It is restricted to unintended
immunosuppression and immunoenhancement, excluding allergenicity or drug-specific
autoimmunity.
This guideline applies to new pharmaceuticals intended for use in humans, as well as to
marketed pharmaceuticals proposed for different indications or other variations on the current
product label in which the change could result in unaddressed and relevant immunotoxicity
issues. In addition, the guideline might also apply to drugs for which clinical signs of
immunotoxicity are observed during clinical trials and following approval to market. The
guideline does not apply to biotechnology-derived pharmaceutical products covered by ICH
S6 Guideline1
and other biologicals.
Existing guidance documents on sensitization or hypersensitivity remain in force and are not
affected by this document. It is beyond the scope of this guideline to provide specific
guidance on how each immunotoxicity study should be performed. General methodology
guidance is provided in the Appendix.
1.4 Overview
The general principles that apply to this guideline are:
1) All new human pharmaceuticals should be evaluated for the potential to produce
immunotoxicity.
2) Methods include standard toxicity studies (STS) and additional immunotoxicity
studies conducted as appropriate. Whether additional immunotoxicity studies are
appropriate should be determined by a weight of evidence review of factor(s) in section
2.1.
The description of the guideline below will follow the recommended decision process in
immunotoxicity evaluation as shown in the flow diagram (Figure 1). More detailed
descriptions of the testing methods are in the Appendix.
2. GUIDELINE
2.1 Factors to Consider in the Evaluation of Potential Immunotoxicity
Factors to consider that might prompt additional immunotoxicity studies can be identified in
the following areas: (1) findings from STS, (2) the pharmacological properties of the drug, (3)
the intended patient population, (4) structural similarities to known immunomodulators,
(5) the disposition of the drug, and (6) clinical information.
The initial screen for potential immunotoxicity involves standard toxicity studies. Data from
rodent and non-rodent studies from early short term to more chronic repeat-dose studies
should be taken into consideration. Additional details on the parameters that should be
evaluated and the reporting of histopathology findings are provided in the Appendix.
© EMEA 2006 5
2.1.1 Standard Toxicity Studies
Data from STS should be evaluated for signs of immunotoxic potential. Signs that should be
taken into consideration are the following:
1) Hematological changes such as leukocytopenia/leukocytosis, granulocytopenia/
granulocytosis, or lymphopenia/ lymphocytosis;
2) Alterations in immune system organ weights and/or histology (e.g. changes in thymus,
spleen, lymph nodes, and/or bone marrow);
3) Changes in serum globulins that occur without a plausible explanation, such as effects
on the liver or kidney, can be an indication that there are changes in serum
immunoglobulins;
4) Increased incidence of infections;
5) Increased occurrence of tumors can be viewed as a sign of immunosuppression in the
absence of other plausible causes such as genotoxicity, hormonal effects, or liver
enzyme induction.
Changes in these parameters could reflect immunosuppression or enhanced activation of the
immune system. Immunosuppression is usually reflected by reduced values of immune
parameters, whereas immunoenhancement is usually reflected by increased values. However,
these relationships are not absolute and can be inverted in some cases.
Similar to the assessment of risk with toxicities in other organ systems, the assessment of
immunotoxicity should include the following:
• Statistical and biological significance of the changes,
• Severity of the effects,
• Dose/exposure relationship,
• Safety factor above the expected clinical dose,
• Treatment duration,
• Number of species and endpoints affected,
• Changes that may occur secondarily to other factors (e.g. stress, see the Appendix,
section 1.4),
• Possible cellular targets and/or mechanism of action,
• Doses which produce these changes in relation to doses which produce other
toxicities and
• Reversibility of effect(s).
2.1.2 Pharmacological Properties
If the pharmacological properties of a test compound indicate it has the potential to affect
immune function (e.g. anti-inflammatory drugs), additional immunotoxicity testing should be
considered. Information obtained from the nonclinical pharmacology studies on the ability of
the compound to affect the immune system could be used in a weight of evidence approach to
decide if additional immunotoxicity studies are needed.
2.1.3 Intended Patient Population
Additional immunotoxicity studies might be warranted if the majority of the patient
population for whom the drug is intended is immunocompromised by a disease state or
concurrent therapy.
© EMEA 2006 6
2.1.4 Structural Similarity
Compounds structurally similar to compounds with known immunosuppressive properties
should also be considered for additional immunotoxicity testing.
2.1.5 Disposition of the Drug
If the compound and/or its metabolites are retained at high concentrations in cells of the
immune system, additional immunotoxicity testing should be considered.
2.1.6 Signs Observed in Clinical Trials or Clinical Use
Clinical findings suggestive of immunotoxicity in patients exposed to the drug could call for
additional nonclinical immunotoxicity testing.
2.2 Weight of Evidence Review
A weight of evidence review should be performed on information from all the factors outlined
above to determine whether a cause for concern exists. A finding of sufficient magnitude in a
single area should trigger additional immunotoxicity studies. Findings from two or more
factors, each one of which would not be sufficient on its own, could trigger additional studies.
If additional immunotoxicity studies are not performed, the sponsor should provide
justification.
3. SELECTION AND DESIGN OF ADDITIONAL IMMUNOTOXICITY STUDIES
3.1 Objectives
If a cause for concern is identified, additional immunotoxicity studies should be performed to
verify the immunotoxic potential of the compound. These studies can also help determine the
cell type affected reversibility, and the mechanism of action. This type of information can
also provide more insight into potential risk and possibly lead to biomarker selection for
clinical studies.
3.2 Selection of assays
If the weight-of-evidence review indicates that additional immunotoxicity studies are called
for, there are a number of assays which can be used. If there are changes in standard toxicity
testing data suggesting immunotoxicity, the type of additional immunotoxicity testing that is
considered appropriate will depend on the nature of the immunological changes observed and
the concerns raised by the class of compound. It is recommended that an immune function
study be conducted, such as a T-cell dependent antibody response (TDAR). If specific cell
types that are affected in STS are not known to participate in a TDAR, assays that measure
function of that specific affected cell type might be conducted (see the Appendix). Where a
specific target is not identified, an immune function study such as the TDAR is
recommended.
In addition, immunophenotyping of leukocyte populations, a non-functional assay, can be
conducted to identify the specific cell populations affected and might provide useful clinical
biomarkers.
© EMEA 2006 7
3.3 Study Design
To assess drug-induced immunotoxicity, a generally accepted study design in rodents is a 28
day study with consecutive daily dosing. Adaptations of immunotoxicity assays have been
described using non-rodent species. The species, strain, dose, duration, and route of
administration used in additional immunotoxicity studies should be consistent, where
possible, with the standard toxicity study in which an adverse immune effect was observed.
Usually both sexes should be used in these studies, excluding nonhuman primates. Rationale
should be given when one sex is used in other species. The high dose should be above the no
observed adverse effect level (NOAEL) but below a level inducing changes secondary to
stress (see Appendix, section 1.4). Multiple dose levels are recommended in order to
determine dose-response relationships and the dose at which no immunotoxicity is observed.
3.4 Evaluation of Additional Immunotoxicity Studies and Need for Further Studies
Results from additional immunotoxicity studies should be evaluated as to whether sufficient
data are available to reasonably determine the risk of immunotoxicity.
1. Additional studies might show that no risk of immunotoxicity can be detected and no
further testing is called for.
2. Additional studies might demonstrate a risk of immunotoxicity but fail to provide
sufficient data to make a reasonable risk-benefit decision. In this case further testing
might help provide sufficient information for the risk-benefit decision.
3. If the overall risk-benefit analysis suggests that the risk of immunotoxicity is
considered acceptable and/or can be addressed in a risk management plan (see ICH
E2E Guideline2
), then no further testing in animals might be called for.
4. TIMING OF IMMUNOTOXICITY TESTING IN RELATION TO CLINICAL STUDIES
If the weight-of-evidence review indicates that additional immunotoxicity studies are
appropriate, these should be completed before exposure of a large population of patients,
usually Phase III. This will allow for the incorporation of monitoring immune system
parameters in the clinical studies if appropriate. The timing of the additional immunotoxicity
testing might be determined by the nature of the effect by the test compound and the type of
clinical testing that would be called for if a positive finding is observed with the additional
immunotoxicity testing. If the target patient population is immunocompromised,
immunotoxicity testing can be initiated at an earlier time point in the development of the drug.
REFERENCES
1. ICH Harmonized Tripartite Guideline (S6) “Preclinical Safety
Evaluation of Biotechnology-Derived Pharmaceuticals”
2. ICH Harmonized Tripartite Guideline (E2E)
“Pharmacovigilance Planning”
© EMEA 2006 8
Figure 1: Flow Diagram for Recommended Immunotoxicity Evaluation
(2.1) Identify factors to consider
(2.2) Weight of evidence (WoE) review
Additional nonclinical
immunotoxicity testing
not needed
NO
(3.0) Conduct additional immunotoxicity studies
YES
WoE review
warrants additional
immunotoxicity
Testing ?
(3.4) Significant
changes
observed?
YES
(3.4 Pt 1) Further
nonclinical
immunotoxicity testing
not needed
NO
All human pharmaceuticals (non-biologicals)
(3.4) Sufficient
data for risk
assessment / risk
management?
NO
YES
(3.4 Pt 2) Consider further immunotoxicity testing
(3.4 Pt 3) Further
nonclinical
immunotoxicity testing
not needed
© EMEA 2006 9
APPENDIX: Methods to Evaluate Immunotoxicity
1. Standard Toxicity Studies
The following table lists the parameters that should be evaluated in standard toxicity studies
for signs of immunotoxicity. These parameters (excluding hematology and clinical chemistry)
and methods for obtaining samples and evaluating tissue sections are described in more detail
in documents from professional toxicological pathology societies.
Parameter Specific Component
Hematology Total and absolute differential leukocyte counts
Clinical Chemistry Globulin levels1
and A/G ratios
Gross pathology Lymphoid organs / tissues
Organ weights Thymus, spleen (optional: lymph nodes)
Histology Thymus, spleen, draining lymph node and at least one
additional lymph node, bone marrow2
, Peyer’s patch3
, BALT4
,
NALT4
1
Unexplained alterations in globulin levels could call for measurement of immunoglobulins.
2
Unexplained alterations in peripheral blood cell lines or histopathologic findings might
suggest that cytologic evaluation of the bone marrow would be appropriate.
3
Oral administration only.
4
For inhalation or nasal route only. BALT: bronchus-associated lymphoid tissues. NALT:
nasal-associated lymphoid tissues
1.1 Hematology and Clinical Chemistry
Total leukocyte counts and absolute differential leukocyte counts are recommended to assess
immunotoxicity. When evaluating changes in globulin levels, other factors should be taken
into account (e.g. liver toxicity, nephrotoxicity). Changes in serum globulins can be an
indication that there are changes in serum immunoglobulins. Although serum
immunoglobulins are an insensitive indicator of immunosuppression, changes in
immunoglobulins levels can be useful in certain situations in order to better understand target
cell populations or mechanism of action.
1.2 Gross Pathology and Organ Weights
All lymphoid tissues should be evaluated for gross changes at necropsy. However, this can be
more difficult for the Peyer’s patches of rodents due to the small size. Spleen and thymus
weights should be recorded. To minimize variability of spleen weights in dogs and monkeys,
bleeding the animals thoroughly at necropsy is recommended. Atrophy of the thymus with
aging can preclude obtaining accurate thymus weight.
1.3 Histopathological Examination
Histopathological changes of the spleen and thymus should be evaluated as an indicator of
systemic immunotoxicity. The lymphoid tissue that drains or contacts the site of drug
administration (and therefore is exposed to the highest concentration of the drug) should be
examined. These sites include the Peyer’s patches and mesenteric lymph nodes for orally
administered drugs, bronchus-associated lymphoid tissues (BALT) for drugs administered by
the inhalation route, nasal-associated lymphoid tissues (NALT) for drugs administered by the
inhalation or nasal route (if possible), and the most proximal regional draining lymph nodes
© EMEA 2006 10
for drugs administered by the dermal, intramuscular, intradermal, intrathecal, or subcutaneous
routes. The specific node selected and the additional lymph node should be at the discretion of
the sponsor based on the sponsor's experience. For intravenously administered drugs, the
spleen can be considered the draining lymphoid tissue.
It is recommended that a “semi-quantitative” description of changes in compartments of
lymphoid tissues be used in recording changes and reporting treatment-related changes in
lymphoid tissues.
1.4 Interpretation of Stress Related Changes
With standard toxicity studies, doses near or at the maximum tolerated dose can result in
changes to the immune system related to stress (e.g. by exaggerated pharmacodynamic
action). These effects on the immune system might be mediated by increased corticosterone
or cortisol release or other mediators. Commonly observed stress-related immune changes
include increases in circulating neutrophils, decreases in circulating lymphocytes, decreases in
thymus weight, decreases in thymic cortical cellularity and associated histopathologic
changes, and changes in spleen and lymph node cellularity. Increases in adrenal gland weight
and/or histologic evidence of adrenal cortical hyperplasia can also be observed. Thymic
weight decreases in the presence of clinical signs, such as decreased body weight and physical
activity, are too often attributed to stress. These findings on their own should not be
considered sufficient evidence of stress-related immunotoxicity. The evidence of stress should
be compelling in order to justify not conducting additional immunotoxicity studies.
2. Additional Immunotoxicity Studies
2.1 Assay Characterization and Validation
In general, the immunotoxicity test selected should be widely used and have been
demonstrated to be adequately sensitive and specific for known immunosuppressive agents.
However, in certain situations, extensive validation might have not been completed and/or the
assay might not be widely used. In these situations, a scientific/mechanistic basis for use of
the assay is called for and, if feasible, appropriate positive controls should be incorporated.
There can be variations of response for each type of immunotoxicity test used by different
labs. In most situations, these changes do not affect the ability of the assay to assess
immunotoxicity. However, to ensure proper assay performance and lab proficiency, several
standard technical validation parameters should be observed. These parameters can include
determining intra- and inter-assay precision, technician-to-technician precision, limit of
quantitation, linear region of quantitation and test sample stability. In addition, assay
sensitivity to known immunosuppressive agents should be established. It is recommended that
each laboratory test a positive control concomitantly with an investigational compound or
periodically in order to demonstrate proficiency of performance, except for studies with nonhuman
primates. For immunophenotyping, if properly validated technically, the addition of
positive controls for each study might not be needed.
Immunotoxicity studies are expected to be performed in compliance with Good Laboratory
Practice (GLP). It is recognized that some specialized assays, such as those described below,
might not comply fully with GLP.
© EMEA 2006 11
2.2 T-cell Dependent Antibody Response (TDAR)
The TDAR should be performed using a recognized T-cell dependent antigen (e.g. sheep red
blood cells (SRBC) or keyhole limpet hemocyanin (KLH)) that results in a robust antibody
response. The endpoint selected should be justified as the most appropriate for the chosen
assay and the selected species.
Antigens for immunization should not be used with adjuvants without justification. Alum
might be considered acceptable for use only in non-human primate studies. The relative
TDAR response can be strain-dependent, especially in mice. With outbred rats, there can be
significant variability among rats within the same group. Inbred rat strains could be used with
provision of sufficient exposure data to bridge to the strain used in the STS.
Antibody can be measured by using an ELISA or other immunoassay methods. One
advantage of this method over the antibody forming cell response is that samples can be
collected serially during the study. In monkeys, serial blood collection can be important due
to the high inter-animal variability in the kinetics of the response. For these studies, data can
be expressed as the sum of the antibody response over several collection dates (e.g. area under
the curve).
When SRBC antigens are used for an ELISA, the preparation of the capture antigen that is
coated on the plates is considered critical. Whole fixed erythrocytes or membrane
preparations can be used as the SRBC capture antigen. ELISA results should be expressed
either as concentration or as titer, but expression as optical densities is not recommended.
2.3 Immunophenotyping
Immunophenotyping is the identification and/or enumeration of leukocyte subsets using
antibodies. Immunophenotyping is usually conducted by flow cytometric analysis or by
immunohistochemistry.
Flow cytometry, when employed to enumerate specific cell populations, is not a functional
assay. However, flow cytometry can be used to measure antigen-specific immune responses
of lymphocytes. Data obtained from peripheral blood can be useful as a bridge for clinical
studies in which peripheral blood leukocytes are also evaluated. It is recommended that
absolute numbers of lymphocyte subsets as well as percentages be used in evaluating
treatment-related changes.
One of the advantages of immunohistochemistry over flow cytometry is that tissues from
standard toxicity studies can be analyzed retrospectively if signs of immunotoxicity are
observed. In addition, changes in cell types within a specific compartment within the
lymphoid tissue can be observed. Some of the lymphocyte markers for certain species are
sensitive to formalin fixation and can only be localized in tissue that are either fixed with
certain fixatives or flash frozen. Quantitation of leukocytes and intensity of staining is much
more difficult with immunohistochemistry.
When immunophenotyping studies are used to characterize or identify alterations in specific
leukocyte populations, the choice of the lymphoid organs and/or peripheral blood to be
evaluated should be based on changes observed. Immunophenotyping can be easily added to
standard repeat dose toxicity studies and changes can be followed during the dosing phase and
periods without drug exposure (reversal period).
© EMEA 2006 12
2.4 Natural Killer Cell Activity Assays
Natural killer (NK) cell activity assays can be conducted if immunophenotyping studies
demonstrate a change in number, or if STS studies demonstrate increased viral infection rates,
or in response to other factors. In general, all NK cell assays are ex vivo assays in which
tissues (e.g. spleen) or blood are obtained from animals that have been treated with the test
compound. Cell preparations are co-incubated with target cells that have been labeled with
51
Cr. New methods that involve non-radioactive labels can be used if adequately validated.
Different effector to target cell ratios should be evaluated for each assay to obtain a sufficient
level of cytotoxicity and generate a curve.
2.5 Host Resistance Studies
Host resistance studies involve challenging groups of mice or rats treated with the different
doses of test compound with varying concentrations of a pathogen (bacteria, fungal, viral,
parasitic) or tumor cells. Infectivity of the pathogens or tumor burden observed in vehicle
versus test compound treated animals is used to determine if the test compound is able to alter
host resistance. Models have been developed to evaluate a wide range of pathogens such as
Listeria monocytogenes, Streptococcus pneumoniae, Candida albicans, influenza virus,
cytomegalovirus, Plasmodium yoelii and Trichinella spiralis. Tumor host resistance models
in mice have used the B16F10 melanoma and PYB6 sarcoma tumor cell lines.
Host resistance assays can provide information on the susceptibility to particular classes of
infectious agents or tumor cells and can have an impact on the risk management plan. In
addition, they can have an important role in identifying or confirming the cell type affected by
a test compound. Moreover, host resistance assays involve innate immune mechanisms for
which specific immune function assays have not been developed. In conducting host
resistance studies, the investigator should carefully consider the direct or indirect (nonimmune
mediated) effects of the test compound on the growth and pathogenicity of the
organism or tumor cell. For instance, compounds that inhibit the proliferation of certain
tumor cells can seem to increase host resistance. An in vitro assay to test direct effects on the
organism is recommended.
2.6 Macrophage/Neutrophil Function
In vitro macrophage and neutrophil function assays (phagocytosis, oxidative burst,
chemotaxis, and cytolytic activity) have been published for several species. These assays
assess macrophage/neutrophil function of cells exposed to the test compound in vitro or
obtained from animals treated with the test compound (ex vivo assay). In vitro exposure to test
compound can also be investigated. An in vivo assay can also be used to assess the effects on
the reticuloendothelial cell to phagocytize radioactively or fluorescently labeled targets.
2.7 Assays to Measure Cell-Mediated Immunity
Assays to measure cell-mediated immunity have not been as well established as those used for
the antibody response. These are in vivo assays where antigens are used for sensitization. The
endpoint is the ability of drugs to modulate the response to challenge. Delayed-type
hypersensitivity (DTH) reactions with protein immunization and challenge have been reported
for mice and rats. Models in which contact sensitizers are used have been explored in mice
but have not been well validated or extensively used. Cytotoxic T cell response can be
© EMEA 2006 13
generated in mice using a virus, tumor cell line, or allograft as the antigenic challenge.
Monkey DTH reactions have also been reported. However, these reactions in monkeys are
very difficult to consistently reproduce. In addition, one should make sure that the DTH
response is not mistaken for an antibody and complement mediated Arthus reaction.