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11
Biological therapies for cancer
Gautham K. Rao
1
, Claudette L. Fuller
2
and Karen D. Price
3
1
Genentech, Department of Safety Assessment, South San Francisco, CA, United States;
2
Non-Clinical Safety and Toxicology, Genmab, Plainsboro, NJ, United States;
3
Janssen,
Research and Development, Springhouse, PA, United States
OUTLINE
Introduction 350
Global regulatory guidances 351
General principles of toxicology
assessments 352
Nonclinical studies and principles of
study design 355
Specialty toxicology assessments 356
Safety pharmacology 356
Developmental and reproductive
toxicology (DART) 356
Genotoxicity and carcinogenicity 358
Immunotoxicity 359
Tissue-binding study 359
Pharmacokinetics (PK), immunogenicity,
and pharmacodynamics (PD) 360
Nonclinical development of marketed
biologics 364
Monoclonal antibodies 366
Rituximab 367
Bevacuzimab 369
Trastuzumab 374
Ipilimumab 377
PD-1 blockade and DAR T 384
Development of relatlimab as a
combination immunotherapy 385
Recombinant analogs of endogenous
human proteins (interferons, cytokines,
enzymes) 387
Roferon-A 388
Summary 390
References 391
Further reading 396
CHAPTER
349
Nonclinical Development of Biologics, Vaccines and
Specialty Biologics, Second Edition
https://doi.org/10.1016/B978-0-443-13344-2.00010-9
© 2025 Elsevier Inc. All rights are reserved, including those for text
and data mining, AI training, and similar technologies.
Introduction
In many cases, conventional treatments for cancerdsuch as surgery, radiation, and chem-
ical chemotherapydhave clinical success, but they also have limitations. These types of treat-
ments are usually designed to target cancer cells directly rather than engaging the body’s
response to the tumor. Surgery and radiotherapy have shown some success in the treatment
of localized tumors, but they may not be useful for disseminated disease or for tumors
located in areas that are difficult or dangerous to reach. Traditional chemotherapy, although
effective, can be severely toxic to normal tissue. Despite the potency of cytotoxic chemo-
therapy and the specificity of immunotherapy, neither method alone has been sufficient to
eradicate the disease. However, there is evidence that standard chemotherapy may work
in synergy with active immunization for more effective antitumor immunity, and there is
great potential for the combination of these treatment modalities (Baxevanis et al., 2009;
Boyle, 2010). Biological therapies can also use the body’s immune system to fight cancer or
to lessen the side-effects that may be caused by some cancer treatments.
The identification of tumor-specific antigens, lymphocytes, and T-cell responses in cancer
patients has led to immunotherapies designed to enhance the antitumor immune response
(Baxevanis et al., 2009). T cells are potent cellular effectors of the immune system and have a
memory that responds if rechallenged by the same antigen (Chiriva-Internat et al., 2008 ).
Since tumor immunotherapy is more specific and less toxic than traditional chemotherapy,
it may be an ideal adjuvant treatment for patients with malignancies that have a high risk of
relapse. Chemotherapy could be used first t o induce the active disease into remission, fol-
lowed by tumor immunotherapy via memory T cells (Chiriva-Internat et al., 2008). Immu-
notherapies seem to be effective against small tumor burdens, and there is increasing
evidence that they can also control large tumor masses to an extent. However, patients
with advanced disease have compromised immunity, which may limit the effectiveness
of immunotherapy. In addition, the redundancy of signaling pathways leading to tumor
cell evasion of the immu ne system can limit the effectiveness of therapies over time. This
has led to the development of novel biological constructs for cancer treatment including bi-
ologics linked to radioisotopes or cytotoxins and bispeci fic antibodies or antibody-like
molecules.
Although the side-effect profile of biologic agents is relatively mild compared with tradi-
tional chemotherapy, biologics have unique issues that must be managed during develop-
ment. A monoclonal antibody (mAb) specific for a target antigen that is expressed on
cancer or autopathogenic cells but also highly expressed on normal cells and involved in
normal cell function (e.g., rituximab, cetuximab, or trastuzumab) is likely to have more po-
tential toxicity than an mAb against an antigen that is either not expressed in humans
(e.g., palivizumab [anti-RSV]), or that has a restricted tissue expression or function (Bria
et al., 2008). Pharmacologic (i.e., target-mediated) toxicities are often observed with biological
therapies and these can often be long-lasting due to the inherent long half-life of many bio-
logics. Thus, nonclinical testing of biopharmaceuticals is often tailored toward ensuring that
pharmacologic toxicities are adequately assessed and, if appropriate, able to be monitored
clinically. Primary tumors usually can be treated with a combination of standard therapies;
however, preventing metastasis through disseminated tumor cells is not always possible.
The goals of cancer immunotherapy are to prevent the disease from spreading and to
11. Biological therapies for cancer350
III. Specialty biologics and indications
improve the patient’s quality of life. Recent studies have suggested that there may be synergy
between immunotherapy and standard chemotherapy; therefore, it may be beneficial to start
biologic therapy earlier in the cancer treatment, before the immune system is compromised
(Baxevanis et al., 2009).
There are many different types of biologics being used and developed for oncology indi-
cations, including mAbs, recombinant proteins, cytokines, growth factors, enzymes, biolog-
ical immunomodulators, radiolabeled biologics, small molecules conjugated to biologics,
hormones and metabolic factors, synthetic peptides, vaccines, oligonucleotides and siRNA.
Based on the properties of these molecules, each can present its own development challenges.
The following sections outline the global regulatory guidelines, considerations for toxicology
assessments, and principles of nonclinical study design for the development of biologics for
oncology indications. In addition, some examples of the nonclinical development of select
marketed oncology biologics is also provided in a subsequent section.
Global regulatory guidances
The nonclinical development of oncology biologics is driven by three international guid-
ances, which when taken together, can be used to derive a toxicology plan for the specific
product of interest. These guidances include ICH M3(R2) Nonclinical Safety Studies for the
Conduct of Human Clinical Trials for Pharmaceuticals (ICH M3, 2009), ICH S6(R1) Preclinical
Safety Evaluation of Biotechnology-derived Pharmaceuticals (ICH S6, 2011), ICH S9 Nonclin-
ical Evaluation of Anticancer Pharmaceuticals, and ICH S9 Questions and Answers (ICH S9,
2010; ICH S9 Q&A, 2018). The M3 guidance attempts to define a general scope or framework
for the required safety studies for all pharmaceuticals, while S6 and S9 narrow the scope and
point out potential differences in requirements for biological agents and anticancer indica-
tions, respectively. The ICH S6 guidance applies to biotechnology-derived pharmaceuticals
and also discusses species selection, study design, immunogenicity and other topics relevant
to the development of biologic therapeutics. This guidance is summarized in detail in Chap-
ter 3. The ICH S9 guidance provides information for pharmaceuticals that are only intended
to treat late-stage or advanced cancer patients, including biotechnology-derived pharmaceu-
ticals, but does not apply to pharmaceuticals intended for patients with long life expectancy,
cancer prevention, treatment of symptoms or side effects of chemotherapeutics, studies in
healthy volunteers, vaccines, or cellular or gene therapy. While ICH S9 provides an overall
guideline for the nonclinical development of oncology therapeutics, this guidance does not
provide a comprehensive list of nonclinical studies that must be conducted prior to entry
into Phase 1 development. Rather a general discussion of the scientific principles in particular
situations is presented (Wang et al., 2010). Therefore, in addition to ICH S9, developers of
oncology biologics should give due consideration to ICH S6 for the design of nonclinical
studies. If healthy volunteers are included in clinical trials, the ICH M3 guideline should
also be followed for those phases of clinical development that apply to healthy volunteers
in addition to ICH S9.
ICH S9 Nonclinical Evaluation for Anticancer Pharmaceuticals: Q&A provides further
clarification of several points discussed in ICH S9 (ICH S9 Q&A, 2018). Many of these are in-
tegrated into this chapter, but key points for advanced cancer biologics include:
Global regulatory guidances 351
III. Specialty biologics and indications
(1) Adefinition of which therapeutics are in scope for advanced cancers (Section 1).
(2) Clarification that if an advanced cancer therapeutic extends survival of patients, no
additional general toxicity studies are usually warranted. In addition, data generated
in patients when the initial progra m was in a refractory late-stage disease should be
considered and may be used to abbreviate the nonclinical program.
(3) Agreement that recovery groups are not automatically warranted in all general
toxicology studies, if scientifically justified and an understanding of reversibility of
findings is presented.
(4) Tissue cross-reactivity studies are not required for the initial or continued clinical
evaluation of advanced cancer therapeutics, unless there is cause for concern.
(5) NHP studies should not be used as default approaches to developmental and
reproductive toxicology risk assessment. If a weight of evidence assessment determines
a reprod uctive hazard, an in vivo study is not warranted.
(6) Combination toxicology studies may not always be required if each of the combination
components has sufficient human safety data (completed Phase 1 or monotherapy
Phase 1) available. In cases where there is no or limited human safety data on one of
the components, a disease model or other in vivo pharmacology study may
supplement toxicology data for the single agents.
(7) Guidance around what is expected for antibody drug conjugates (see Chapter 6,
Nonclinical Development of ADCs) regarding toxicology study and genetic toxicology
considerations.
All of the guidances delineate the objectives of the nonclinical evaluations as: (1) the iden-
tification of the pharmacologic properties of a pharmaceutical; (2) the establishment of a safe
initial dose level for the first human exposure; and (3) the establishment and understanding
of the toxicological profile of a pharmaceutical including target organs, estimation of safety
margin, and reversibility of any toxicities.
As mentioned in ICH M3 (ICH M3, 2009):
pharmaceuticals under development for indications in life-threatening or serious diseases without current
effective therapy may warrant a case-by-case approach to both the toxicological evaluation and clinical
development to optimize and expedite drug development. In these cases, particular studies may be abbre-
viated, deferred, or omitted.
In clinical studies of cancer patients, the dose levels may be close to or at exposure levels at
which adverse effects have been demonstrated. For these reasons, the type and timing and
flexibility called for in designing nonclinical studies of anticancer pharmaceuticals may be
different from other pharmaceuticals. Therefore, while the ICH guidances in combination
provide overall requirements, the approach to nonclinical study design, conduct and end-
points to be included for biologics must be driven by scientific rationale.
General principles of toxicology assessments
The most important consideration for nonclinical studies is the selection of relevant spe-
cies; that is, one in which the test material is pharmacologically active due to the expression
11. Biological therapies for cancer352
III. Specialty biologics and indications
of the receptor or an epitope (e.g., in the case of mAbs). In accordance with the principles out-
lined in ICH S6(R1), safety studies should be conducted in two nonclinical species (rodent
and nonrodent), if both are determined to be relevant. The biological activity together with
species and/or tissue specificity of many biotechnology-derived pharmaceuticals often pre-
clude standard toxicity testing designs in commonly used species (e.g., rats and dogs).
Nonhuman primates are usually used for safety evaluations of biopharmaceuticals when
cross-reactivity can be demonstrated (O’Conner, 2009; Tibbitts et al., 2010). Therefore, a clear
determination of pharmacological activity via in vitro binding and activity assays should pre-
cede the selection of appropriate test species (O’Conner, 2009). In vitro studies may also
contribute significantly to nonclinical dose selection and regimen. Differences in binding af-
finity to target or downstream efficacy between nonclinical species and humans may neces-
sitate adjustment of dose or dose frequency in nonclinical studies for an adequate assessment
of safety. In the absence of pharmacological activity in rodents and dogs, the use of a single
nonhuman primate (NHP) species for safety studies is justifiable. When a biologic therapeutic
is active only in NHPs, the use of cynomolgus monkeys is recommended for use in toxicology
studies over rhesus monkeys. The main reason for preference of cynomolgus monkeys over
the rhesus is because cynomolgus monkeys are nonseasonal breeders, and therefore more
appropriate for use in reproductive studies (Bussiere, 2008), which are required during sub-
mission of the marketing application.
If a biologic therapeutic is active only in humans and chimpanzees, a full in vivo safety
assessment in chimpanzees is not possible because terminal studies in chimpanzees are either
not permitted or banned (O’Conner, 2009; Tibbitts et al., 2010). In these cases, a justification of
safety assessment may be feasible through a battery of in vitro human studies combined with
in vivo safety data in transgenic rodents. For example, the in vivo safety assessment of Kelix-
imab, an anti-human CD4 mAb, which is otherwise active only in humans and chimpanzees,
was conducted in transgenic mice expressing human CD4 (Bugelski et al., 2000). However,
transgenic animal models should be well characterized with respect to the cellular and
biochemical interactions of the human receptor within the model species as well as down-
stream signaling events that occur following engagement of the human receptor. Transgenic
animals should show similar physiological consequences following the interaction of the bio-
logic product and the humanized receptor or ligand and be established as a relevant species
for nonclinical safety studies. As stated in the ICH S6(R1) guidance “toxicity studies in non-
relevant species may be misleading and are discouraged.”
Useful information may also be gained from studies of homologous proteins or surrogate
proteins active in rodents. However, it should be noted that the production process, range of
impurities/contaminants, pharmacokinetics, and exact pharmacological mechanism(s) may
differ between the homologous form and the product intended for clinical use and could
affect data interpretation. Therefore, homologs and surrogate equivalents may not allow
for a nonclinical safety assessment of the clinical candidate itself. In the absence of transgenic
animal models or homologous proteins or if these models may not be relevant, it may still be
prudent to assess some aspects of potential toxicity in a limited evaluation in a single species,
for example a repeated-dose toxicity study of <14 days duration that includes an evaluation
of important functional endpoints (e.g., cardiovascular and respiratory). The inclusion of rele-
vant in vitro tests relating to safety using human cells or cell lines is also encouraged and can
inform on certain risks pertaining to patients.
General principles of toxicology assessments 353
III. Specialty biologics and indications
In the case of certain types of targeted cytotoxic drugs (i.e., a cytotoxic molecule linked to a
mAb or targeted fusion protein directed against a receptor or ligand on the cell type of inter -
est), additional studies may be performed in other relevant species to evaluate or further
characterize the properties of the cytotoxic agent. In species where the biological portion of
the pharmaceutical does not cross-react, the lack of targeting of the cytotoxic agent will
need to be factored into the overall profile as well as potential differences in systemic expo-
sure and tissue half-life. However, in the case of biologics conjugated to cytotoxics, the toxi-
cologic profile of the cytotoxic agent is often already known and can be referenced.
Prior to Phase 1 studies, preliminary characterization of the mechanism(s) of action, resis-
tance, and schedule dependencies as well as antitumor activity should be conducted. Appro-
priate models should be selected based on the target and mechanism of action but need not
be studied using the same tumor types intended for clinical evaluation (Grimaldi et al., 2023).
These studies can provide nonclinical proof of principle, guide schedules and dose escalation
schemes, provide information for selection of test species, aid in starting dose selection, and
in some cases justify pharmaceutical combination where clini cal information cannot be ob-
tained. Secondary pharmacodynamic or off-target effects should be investigated as
appropriate.
The physiological state of the animals may also be a key factor in the design of nonclinical
studies with biologics. In general, nonclinical toxicology studies are conducted in normal
healthy animals. However, when the target protein is either not expressed or under
expressed, the use of healthy animals for toxicology studies may not be appropriate. The
use of animal models of human disease may be acceptable as an alternative to normal healthy
animals. Animal models of human disease could be spontaneous or induced, and most often
include knockout or transgenic mice. Higher or preferential expression of target protein in
diseased animal models provides the potential for a clearer understanding of pharmaco ki-
netics, improved capacity to perform pharmacodynamic and efficacy assays, and greater rele-
vance to the clinical disease situation. Given that the toxicity of mAbs and other biologics
generally arises from interactions with target antigens rather than off-target effects (Tabrizi
and Roskos, 2007), use of animal models of human disease may translate to a more thorough
assessment of the safety profile. In addition, if target-mediated clearance influences exposure
of the biologic, disease animal models may be more useful than normal animals in under-
standing overall exposure-mediated toxicity (Baumann, 2008). Major disadvantages of this
approach, however, are that nonclinical models of human disease may not exist, and the
value of rodent models may be diminished as a result of antidrug antibody (ADA) respo nse
to the human biologics. Further, the disease state of the animals may confound the interpre-
tation of toxicity data.
Additional considerations for design of nonclinical studies with biologics include the age
of the animals, route of administration, and treatment regimen. In general, toxicology studies
for oncology therapeutics should use adult animals of both sexes. However, based on the
indication and incidence among humans, use of adult animals of a single sex may be desir-
able, and should be thoroughly justified. According to ICH S9, toxicology studies in juvenile
animals are not required in order to include pediatric populations in trials for oncology indi-
cations and should only be considered when human safety data and previous animal studies
are considered insufficient for a safety evaluation. The frequency and route of administration
of biologic therapeutics should generally be similar to that intended for the proposed clinical
11. Biological therapies for cancer354
III. Specialty biologics and indications
trial (DeGeorge et al., 1998). Frequency of administration in nonclinical safety studies may be
modified to account for differences in absorption or clearance rates in laboratory animals. In
addition, in order to accommodate different dosing schedules that might be utilized in initial
clinical trials, a higher dosing frequency or multiple routes of administration may be
explored.
Nonclinical studies and principles of study design
As emphasized in ICH S9, the determination of a no-observed-adverse-effect level
(NOAEL) or no-effect level (NOEL) in toxicology studies is not considered essential to sup-
port a Phase 1 clinical trial in patients with advanced cancer. Toxicology studies should be
designed to support the clinical dosing schedule, which for an oncology biologic with a
long half-life such as many mAbs, can consist of doses spaced apart by as much as 3e4 weeks
or more. In that case, Phase 1 studies can be supported by single-dose or short-term (i.e., less
than 2-week) repeat-dose studies. Prior to the initiation of single-dose or short-term repeat-
dose studies, it is anticipated that pharmacology studies have been conducted to demonstrate
target binding and affinity, nonclinical proof of princip le (e.g., antitumor activity/tumor
regression in mouse models), and relevance of the test species. Single-dose or short-term
repeat-dose studies should be designed to assess pharmacokinetic (PK) parameters including
C
max
(peak serum levels), area under the curve (AUC; overall exposure) and half-life, should
incorporate standard toxicology parameters (clinical observations, body weights, etc.), clin-
ical pathology parameters (hematology, serum chemistry, coagulation, urinalysis), and
monitor for signs of local irritation at the site of parenteral dose administration. In addition,
since most human proteins are inherently immunogenic in nonclinical species and can result
in neutralizing or clearing antibodies (Tabrizi and Roskos, 2007), evaluation of ADAs may
assist in both the interpretation of study results and in the design of subsequent longer
term toxic ology studies.
According to ICH S9 recommendations, 4-week repeat-dose nonclinical studies should be
conducted to support clinical dosing up to and beyond 4 weeks in Phase I. Moreover, data
from 4-week repeat-dose studies in conjunction with data from Phase I also allows for entry
into Phase II trials. The dosing frequency in the 4-week repeat-dose study should match or
exceed that to be used in the planned clinical trials, as appropriate, with consideration for
any differences in pharmacokinetics in the test species. Apart from standard toxicology, tox-
icokinetics, clinical and anatomic pathology parameters, and immunogenicity (as appro-
priate), 4-week repeat-dose studies should incorporate safety pharmacology endpoints
(cardiovascular, respiratory, and CNS assessments) since stand-alone safety pharmacology
studies are not recommended for biologics by either ICH S9 or ICH S6(R1). Moreover, since
the most common off-target toxicities observed with biologic therapeutics are localized injec-
tion site reactions, injection sites should also be routinely collected and analyzed histopath-
ologically (Haller et al., 2008).
To support clinical trials, at least one repeat-dose nonclinical study should incorporate a
recovery period at the end of the study to assess for reversibility of toxicity findings and
for the potential that toxicity continues to progress after cessation of drug treatment. While
the demonstration of complete recovery from observed toxicities is not considered essential,
Nonclinical studies and principles of study design 355
III. Specialty biologics and indications
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