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Pharmacokinetics and pharmacodynamics assessments
For biopharmaceutical drugs, pharmacokinetics (PK) assessments in toxicology studies
enable comparison of dose/exposure and toxicity relationships and are important in defining
safety margins for human clinical studies. Unlike other biopharmaceutical drugs, vaccines are
administered for a limited period of time and the amount of total antigen in the vaccine is
usually very small compared with the am ount of drug administered for a biopharmaceutical
drug. Pharmacokinetics evaluations for a vaccine antigen are not feasible, since the antigen is
rapidly engulfed by antigen-presenting cells, which process and present the antigen to initiate
the immune response. Pharmacokinetics studies with novel adjuvants are not required but
may help understand the mechanism of action of the adjuvant (Green and Al-Humadi, 2017).
Pharmacodynamic (PD) assessments in toxicology studies with biopharmaceutical dr ugs
provide confirmation that the drug is altering the intended target and therefore any “on-
target” toxicity would be observed. For vaccines, the immune response to the vaccine antigen
can be measured and provides an indication of the PD for the vaccine. Immune responses are
evaluated during the toxicology study to confirm the relevance of the animal model for toxi-
cological evaluation and allow correlations to be made between observed toxicity and the
induced immune response (Green and Al-Humadi, 2017).
This is particularly important in developmen tal and reproductive toxicology studies,
where it can be used to demo nstrate that the fetus/offspring was exposed to maternal anti-
bodies. The assays that are used to assess immunogenicity in nonclinical studies are further
described in this chapter. Detailed evaluation of the PD or immunogenicity of the vaccine is
usually performed in pilot studies prior to the start of toxicology studies in order to obtain
“proof of concept” in animals and select the most appropriate vaccine formulation and
dose level to test in nonclinical and clinical studies.
Assays
Assays used to measure the PK of biopharmaceutical drugs and PD of vaccines have some
similarities. Ligand-binding assays are frequently used, since the biopharmaceutical drug
product and the antibodies elicited by the biopharmaceutical product or vaccine can be
detected using ligand-binding assays. Assays are developed, optimized, and validated or
qualified for use in nonclinical studies. This inc ludes determination of assay sensitivity, selec-
tivity, precision, reproducibility, ruggedness, and quality control specifications (FDA, 2018;
ICH, 2022). The same assay format is often used in nonclinical and clinical studies, with assay
validation or qualification being performed for each different species matrix.
Assays supporting PK assessments of other biopharmaceutical drugs need to be vali-
dated according to a prespecified validation plan and several considerations need to be
taken into account (ICH, 2022). Assays supporting PD assessments of vaccines are
typically qualified and a “fit-for-purpose” assay is utilized to support the nonclinical
studies.
8. Special considerations for the nonclinical safety assessment of vaccines246
II. Vaccines
Assays to detect the PD of vaccines are designed depending on the type of immune
response induced by the vaccine, and the evaluation might include assessment of seroconver-
sion rates, geometric mean antibody titers, or cell-based immune responses. In some cases,
functional immune responses (e.g., neutralizing antibodies) can be evaluated using specific
assay formats. Although animal models have limitations and may not predict immunoge-
nicity in humans, these studies are important to obtain an early “proof-of-concept” evalua-
tion nonclinically and are also included in toxicology and clinical studies. Therefore, the
assays that are used to assess immunogenicity need to be carefully designed. Combi nation
and multivalent vaccines pose a challenge for assay development. Some of the multivalent
vaccines contain antigens that are similar to each other, resulting in the potential for immune
interference in the assays. Therefore, reagent development and selection for specificity is an
important part of assay development.
Ligand-binding assays
Antigen-specific antibody responses following the administration of a vaccine candidate
are measured using ligand-binding assays. Standard Enzyme-Linked Immuno-Sorbent As-
says (ELISAs) are often used to measure antibody responses. In the simplest assay format,
the antigen of interest is coated on the wells of a microtiter plate, serum taken from the dosed
animal is placed in the wells, followed by addition of a species-specific antibody conjugated
to an enzyme, which then produces a colorimetric readout upon addition of a substrate.
For vaccines containing multiple antigens, multiplex technologies allow the simultaneous
evaluation of multiple antigen-specific antibody responses in a single sample. These multi-
plex assays often require substantially less sample and reagents than the traditional ELISA
(which is limited by its ability to measure only a single antigen). Assay platforms include
bead-based (Luminex and Bio-Plex) and multiarrays (Meso Scale Discovery).
Ligand-binding assays are also used to detect biopharmaceutical drugs in the plasma or
serum of dosed animals (for the PK assessment) and the antidrug antibodies that may
form in animals after dosing with the biopharmaceutical drug.
Cell-mediated immune response assays
For vaccines targeting cellular immunity, enzyme-linked immunosorbent spot (ELISpot)
assays can be developed to quantify cytokine-secreting antigen-specific T lymphocytes. The
IFNg ELISpot assay is commonly used as an efficient and sensitive approach to assess the
T-cell respo nse to one or several antigens. In this assay, peripheral blood mononuclear cells
(PBMCs) collected from animals administered the candidate vaccine, are stimulated ex vivo
with the corresponding peptide antigens and the number of IFNg-secreting T lymphocytes is
evaluated using the ELISpot assay. This type of assay has been implemented for vaccines in
which eliciting CD8þ cytotoxic T-lymphocyte (CTL) responses are critical.
Cell-mediated immune response assays 247
II. Vaccines
Functional assays
Functional antibody assays can be divided into three main categories: neutralization,
serum bactericidal antibody (SBA), and opsonopha gocytic assays (OPA). These assays are
more complex than the antibody-based assays described previously, since they include
many biological components, which are difficult to standardize (Feavers and Walker, 2010).
In neutralization assays, antibodies elicited by vaccination are tested for their ability to
block the activity of the antigen, either by binding to an active site on the antigen or blocking
a domain recognized by its receptor on susceptible cells. Neutralization assays are used when
components of the vaccine include toxins (Feavers and Walker, 2010).
SBA assays measure the level of antibody in serum required to kill bacteria by complement
activation. A suspension of bacteria is treated with dilutions of (heat-inactivated) antiserum
followed by the addition of complement. The viability of the bacterial suspension is assessed
at each serum dilution and the bactericidal antibody titer is usually expressed as the dilution
of antiserum that kills a defined proportion of bacteria (Feavers and Walker, 2010).
OPA assays measure the amount of serum antibody required to opsonize bacteria in the
presence of complement and thereby facilitate phagocytosis. In the assay, a bacterial suspen-
sion is treated with dilutions of the test serum, then complement is added followed by a sus-
pension of phagocytic cells. The degree of opsonophagocytosis is determined from the
number of bacteria either killed or taken up by the phagocytes (Feavers and Walker, 2010).
Because of the complexity of these assays, they are not routinely included in GLP-
compliant toxicology studies. They can be used in stand-alone immunogenicity studies
that are performed as part of the “proof-of-concept” nonclinical studies in animals.
Differences in the nonclinical safety assessment of vaccines and
biopharmaceutical drugs
One major difference between the nonclinical safety assessment of vaccines compared
with other biopharmaceutical drugs is that many vaccines in development are prophylactic
vaccines, which are intended to be administered to large numbers of healthy people
including healthy infants and children, in contrast to other biopharmaceutical drugs that
are developed primarily to treat ill patients. This places a significant emphasis on assessing
vaccine safety nonclinically and clinically. Furthermore, the overall incidence of the disease
thatavaccinecandidateisintendedtotreatmaybelow,therefore,alargepercentageof
vaccinated individuals may never benefit from the vaccine themselves. Therefore, it is crit-
ical to adequately evaluate the safety of the vaccine candidates during development (Sut-
kowski and Gruber, 2003).
There are several additional differences between the toxicology studies for vaccines and
other biopharmaceutical drugs. In general, vaccine toxicology programs involve fewer
studies; however, the manufacturing process optimization and release of vaccine material
to be used in these studies require significant time and development efforts. One
8. Special considerations for the nonclinical safety assessment of vaccines248
II. Vaccines
repeat-dose toxicity study is often sufficient for vaccines since the purpose of the study is to
match or exceed the total number of doses that will be administered to humans, while for
other biopharmaceutical drugs more than one repeat-dose toxicity is typically performed;
for example, short-duration toxicology studies support initial clinical studies and long-
duration (chronic) studies support chronic administration in humans. A single species is
typically used for vaccine toxicology studies, whil e one or two species will be used for other
biopharmaceutical products based on cross-reactivity.
Species selection for vaccine toxicology studies is based on demonstration of immune
response induction in the chosen species, while species for toxicolo gy studies with other
biopharmaceutical drugs are selected based on cross-reactivity or pharmacological activity.
The species utilized in toxicology studies also differs between vaccines and other biophar-
maceutical drugs, since rabbits or rodents (rats or mice) are typically used for vaccine toxi-
cology studies while nonhuman primates are often used in addition to rodents for studies
with other biopharmaceutical drugs (particularly with monoclonal antibodies). The study
design, route of administration, and PK/PD assessments also differ. For vaccines, the
full-human dose equivalent must be assessed in the toxicology study and it may not be crit-
ical to evaluate a doseeresponse relationship. In contrast, the doseeresponse relationship
of other biopharmaceutical drugs must be assessed in the repeat-dose toxicity study to
provide adeq uate data for risk assessment and safety evaluation; therefore, multiple dose
levels are typically evaluated in repeat-dose toxicity studies with other biopharmaceutical
drugs.
Safety margin calculations for vaccines are usually expressed as the fold excess by body
weight between humans and the animal species at the NOAEL in the toxicology study since
vaccines are generally given to animals at a fixed dose regardless of the animal body weight
(Wolf et al., 2010). In contrast, safety margins for other biopharmaceutical drugs are calcu-
lated based on the exposure in animals at the NOAEL in the toxicology study compared
with the predicted exposure in humans.
The immunoassays used to support vaccine toxicology studies are for the purpose of con-
firming the vaccine-induced immune response (which is a surrogate for PK and PD), while
the immunoassays used to support toxicology studies with other biopharmaceutical drugs
are used for the purpose of evaluating drug levels (PK), or target engagement (PD) or eval-
uation of antidrug antibodies. Some vaccines require evaluation in specific study types; for
example, biodistribution and integration studies are performed for DNA vaccines, and neuro-
virulence studies are performed for live virus vaccines with neurotropic potential (Green and
Al-Humadi, 2017; Wolf et al., 2010, 2013).
Genotoxicity and carcinogenicity studies are generally not performed for vaccines, while
they might be needed for other types of biopharmaceutical drugs. Reproductive toxicity studies
of vaccines are only required for vaccines intended for women of childbearing potential
(WOCBP). Since many vaccines are intended for infants only, reproductive toxicity studies
may not be needed. Separate safety pharmacology studies are not usually performed for vac-
cines. If needed, evaluations of body temperature, electrocardiogram, and central nervous
system are generally included in the repeat-dose toxicity study (Green and Al-Humadi, 2017).
Differences in the nonclinical safety assessment of vaccines and biopharmaceutical drugs 249
II. Vaccines
Summary
Nonclinical toxicology studies are an important part of the safety evaluation of vaccines.
Early de-risking strategies are important for vaccines and include several considerations
including toxicity, allergenicity, reactogenicity, pyrogenicity, in silico evaluations, and utiliz-
ing animal models of disease if specific concerns need to be addressed. The nonclinical toxi-
cology approach for vaccines shares some similarities with the approach taken with other
biopharmaceuticals. One of the main similarities is that a case-by-case approach needs to
be taken, including careful consideration of the formulation components and animal models
available to address specific concerns. The complexity of vacci ne formulations needs to be
taken into account when developing the nonclinical safety assessment strategy, including
novel excipients and adjuvants. Overall differences in repeat-dose toxicity study designs
for vaccines compared with other biopharmaceutical drugs include: (1) the number and
type of studies required; (2) the routine use of a single species for vaccine toxicology studies,
when compared with one or two species for other biopharmaceutical drugs; (3) the manner in
which species are selected (4) accepted use of a single (i.e., human equivalent); dose level in
vaccine studies, when compared with multiple dose levels in studies with other biopharma-
ceutical drugs; and (5) PK and PD are not measured in a traditional manner as for other bio-
pharmaceuticals but rather the expected immune response is used as a surrogate for these
endpoints in vaccine studies. The immunoassays included in vaccine toxicology studies or pi-
lot immunogenicity studies are carefully developed depending on the type of immune
response being induced by the vaccine. The nonclinical safety assessment of vaccines has
become an important part of the safety evaluation during product development, and pro-
vides a strong foundation from which the overall safety profile of a vaccine is determined.
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Specialty biologics and
indications
SECTION III
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9
Preclinical development of viral
vector gene therapies
Mary S. Newman
1
, Eloise Hudry
2
, Jacqueline Brassard
3
and
Justine J. Cunningham
4
1
Alchemy Development Partners, LLC, San Geronimo, CA, United States;
2
Novartis Institutes
for BioMedical Research, Cambridge, MA, United States;
3
Brassard Toxicologic Pathology
Consultancy Corporation, Tust in, CA, United States;
4
REGENXBIO, Rockville, MD, United
States
OUTLINE
Background: Vectors and applications 258
Delivering gene therapy products:
Nonviral and viral vectors 258
Nonviral vectors 261
Viral vectors 262
Nonintegrating viral vectors 263
Integrating viral vectors 266
Therapeutic applications and beyond 268
General considerations regarding
chemistry manufacturing and controls
(CMC): Viral vectors manufacturing,
characterization, and testing 270
Early decisions regarding CMC 270
Vector development 270
Producer cell line selection (upstream
process) 270
Product format and formulation
(downstream process) 271
Product characterization and potency
assays 272
Product characterization e quality
attributes 272
Potency assays 273
Nonclinical development 273
Principles of nonclinical development 273
Selection of test article for
nonclinical studies 273
Justification for route and method of
administration for nonclinical
studies 274
Selection of animal model and or
species for nonclinical efficacy
studies 274
Pharmacologic efficacy assessments 275
Study design, dose selection and
justification of endpoints 275
CHAPTER
255
Nonclinical Development of Biologics, Vaccines and
Specialty Biologics, Second Edition
https://doi.org/10.1016/B978-0-443-13344-2.00003-1
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