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administration in the early and late gestational studies, respectively). However, an increase in
fetal: maternal serum concentration ratio is consistent with an increase in immunoglobulin
transfer rate observed as gestation progresses in both humans and in NHPs. Compa red
with serum concentrations, trastuzumab was detected at relatively low levels in the milk
of lactating monkeys and had no effect on neonatal growth and development from birth to
1 month of age when the study was terminated.
Since the nonclinical program for trastuzumab preceded the implementation of ICH guid-
ances S2, S6 and S9, its genotoxic potential was investigated both in vitro and in vivo, despite
being a biologic. In vitro studies included an Ames test in Salmonella typhimurium (strains TA
98, 100, 1535, and 1537) and E. coli (strains WP2pKM101 and WP2uvrApKM101), and a chro-
mosome aberration assay in human peripheral lymphocytes. Concentrations up to 5 mg/mL
were employed in both assays, with and without metabolic activation. In an in vivo muta-
genic assay (the micronucleus assay), no evidence of chromosomal damage to mouse bone
marrow cells was observed following bolus intravenous doses of up to 118 mg/kg trastuzu-
mab. All tests demonstrated clearly negative results. Trastuzumab was not tested for its carci-
nogenic potential (Yuan et al., 1996; Hu et al., 2002).
Other toxicity studies performed with trastuzumab included: drug interaction studies,
local tolerance studies, in vitro hemolytic potential/blood compatibility studies, and a sys-
temic toxicity study in mice with the formulation component trehalose. Although PK drug
interaction studies are usually not performed for biologics, a study was undertaken in groups
of female rhesus monkeys to investigate kinetic interactions between trastuzumab and a
range of conventional antitumor drugs (taxol, adriamycin, adriamycin/cytoxan combination)
(Yuan et al., 1996; Hu et al., 2002). The kinetic parameters of the various chemotherapeutics
were essentially unaffected by the presence of trastuzumab and vice versa, except in the case
of the combination with Taxol, where the C
max
for trastuzumab was doubled and the clear-
ance halved, terminal half-life being unaffected. There were no significant findin gs on param-
eters such as mortality and clinical observations, body weights, electrocardiograms, clinical
pathology including hematology, serum chemistry, and urinalysis in these studies. No local
irritation was observed when trastuzumab and trastuzumab excipient were given by single-
bolus IV injection of liquid or lyophilized trastuzumab at a concentration of 5 mg/mL.
Trastuzumab (at a concentration of 5 mg/mL) and trastuzumab vehicle (diluted to a concen-
tration equivalent to a 5 mg/mL trastuzumab concentration) did not cause hemolysis of rhe-
sus monkey or human erythrocytes and are compatible with rhesus monkey and human
serum and plasma. Trehalose, a component of the lyophilized formulation, produced no ev-
idence of clinical or anatomical toxicity when given daily to mice at intravenous doses of up
to 1 g/kg.
In addition, specialized nonclinical studies were conducted in an attempt to elucidate the
mechanism for the enhanced cardiotox icity observed in some clinical trial patients receiving
trastuzumab in combination with anthracycline-based cytotoxics such as doxorubicin (Yuan
et al., 1996; Hu et al., 2002). Single-dose studies in rhesus monkeys with the trastuzumabe
doxorubicin combination (both at 1.5 mg/kg) had previously shown no evidence for cardiac
effects, and tissue cross-reactivity studies with trastuzumab in monkey and human tissue did
not reveal localization to heart tissue. Regardless, regulatory advice apparently obtained at or
about the time of the marketing application submission indicated that the mode of action and
impact of trastuzumab on the enhanced cardiotoxicity of the combination should be studied.
11. Biological therapies for cancer376
III. Specialty biologics and indications
Enhanced cardiotoxicity was not observed in a rat model of doxorubicin cardiotoxicity
following addition of a surrogate antibody specific for rat c-erbB2. Potential models of
anthracycline-induced cardiotoxicity in mice and dogs using trastuzamab were unsuitable
due to species specificity of trastuzumab and in consideration of potential immunogenic re-
sponses to a humanized protein. Possible anthracycline models in the monkey were consid-
ered unsuitable based on ill-defined dose requirements to produce cardiotoxicity (Yuan et al.,
1996; Hu et al., 2002).
Bridging studies conducted in monkeys to evaluate the safety and pharmacokinetics of
trastuzumab, to evalu ate optimization of the manufacturing process including a cell line
change revealed no evidence of acute toxicity or changes in pharmacokinetic disposition in
monkeys. Trastuzumab produced from a subsequent manufacturing scale up and formula-
tion change (lyophilization) resulted in comparable pharmacokinetic profiles in monkeys
and had no effect on safety endpoints (Yuan et al., 1996; Hu et al., 2002).
Ipilimumab
Ipilimumab (Yervoy, MDX-010, BMS-734016) represents a new class of selective T-cell po-
tentiators for the treatment of cancer. It is a fully human IgG1k monoclonal antibody that
blocks the interaction between CTLA-4, expressed on activated T cells, and B7.1 (CD80)
and B7.2 (CD86) molecules, expressed on antigen-presenting cells (APCs), with subsequent
blockade of the inhibitory modulation of T-cell activation promoted by binding of CTLA-4
with B7.1 or B7.2. Adaptive immunity, mediated by tumor-specific T cells, is recognized as
one of the major arms for the immune system to combat tumor progression; thus, augmen-
tation of antitumor T-cell responses represents a novel approach for the treatment of cancer.
The use of ipilimumab for the treatment of human cancer is supported based on the role of
T cells in tumor immunity, the central function of CTLA-4 in negatively regulating T-cell re-
sponses, and the large body of data demonstrating activity of CTLA-4 mAbs in nonclinical
tumor models.
Generation of effector T cells requires the orchestration of multiple signals that control the
magnitude and durability of the response. CD28 costimulation is required for the full activa-
tion of naïve and antigen-experienced T cells. Binding of CD28 to B7.1 or B7.2 on APC results
in increased T-cell proliferation, cytokine production and survival of T cells. Other receptors
then come into play to regulate T-cell responses. The role of CTLA-4 as a key negative regu-
lator of T-cell responses is well established. Once T cells are activated, CTLA-4, located intra-
cellularly in resting T cells other than regulatory T cells (Tregs), is mobilized to the cell
membrane and directly competes with CD28 for binding to B7.1 and B7.2. Since CTLA-4
shows higher affinity to these ligands than CD28, low CTLA4 expression is sufficient to
out-compete CD28.
In vivo studies demonstrated that blockade of CTLA-4 by ipilimumab resulted in
enhanced antigen-specific antibody production to T celledependent antigens in NHPs and
humans and augmented proliferation of T cells against simian immunodeficiency virus
(SIV) DNA vaccines, likely due to unrestricted CD28 costimulation and the removal of inhib-
itory signals elicited by CTLA-4 engagement with the B7.1 and B7.2 molecules. In mouse tu-
mor models, treatment with CTLA-4-blocking mAbs resulted in the induction of an
antitumor immune response, which delayed tumor growth or eradicated established tumors.
Bevacuzimab 377
III. Specialty biologics and indications
In tumor models where anti-CTLA-4 therapy was ineffective, combination with several ther-
apeutic modalities, including surgery, vaccination, radiotherapy, and immunomodulatory
agents, demonstrated synergistic effects to control tumor growth. Treatment with anti-
CTLA-4 mAb is also associated with the development of responses to self-antigens in mouse
models of autoimmunity.
Ipilimumab binds to human and cynomolgus monkey CTLA-4 with high affinity (monkey
with two to four times lower affinity) (EMA, 2011). In vitro, ipilimumab did not elicit
complement-dependent cytotoxicity (CDC), but it demonstrated stronger binding to immobi-
lized IgG Fc receptor I (FcgRI) than FcgRIIA or FcgRIII, thereby showing low to moderate
potential to produce ADCC fo r cells expressing the target (CTLA-4). However, data from
nonclinical and clini cal studies demonstrated that treatment with ipilimumab did not deplete
cell types that express CTLA-4 (i.e., activated T cells or Tregs), and, thus did not exhibit
appreciable ADCC in vivo (Ryan et al., 1999).
The toxicity of ipilimumab was well characterized in a comprehensive toxicology program
that was compliant with GLP regulations. Ipilimumab is pharmacologically active in cyno-
molgus monkeys, the toxicology species utilized, and was evaluated at doses up to 10-fold
greater and dosing-intervals up to seven times more frequent than the current human
dose/regimen. Rodent studies were not performed due to lack of cross-reactivity of ipilimu-
mab to these species. Pivotal single-dose studies with ipilimumab were not conducted since
the initial doses in repeat-dose studies were considered adequate to characterize its acute
toxicity. No acute to xicity was noted in monkeys following the first administration of ipilimu-
mab at doses up to 30 mg/kg in the repeat-dose toxicology studies (Ryan et al., 1999).
The receptor- and tissue-binding and pharmacokinetic characteristics of ipilimumab in
monkeys were comparable with those in humans, indicating the suitability of this species
for the toxicological assessment. In both monkeys and humans, ipilimumab is a long half-
life, low-clearance drug that remains mainly in the vascular system. Therefore, evaluation
of similar dosing frequency in some of the monkey toxicology studies as compared with
humans was considered acceptable. Ip ilimumab generally was not immunogenic in mon-
keys, although 12% of animals did exhibit some degree of an antiipilimumab antibody
response. In some cases, the presence of antidrug antibodies correlated with lower circulating
levels of ipilimumab (Hu et al., 2002).
In NHP studies, IV administration of ipilimumab was tolerated without adverse effects at
doses up to 30 mg/kg per day administered every 3 days for three doses, at 10 mg/kg
administered weekly for 1 month, at 1 mg/kg administered weekly for 10 weeks, and at
doses up to 10 mg/kg/day administered approximately monthly for up to 6 months (Ryan
et al., 1999). In the pivotal 6-month toxicity study, drug-related findings were limited to de-
creases in absolute and relative thyroid and testicular weights; however, there were no cor-
responding microscopic changes in these organs, and thus 10 mg/kg was considered the
NOAEL. Evidence of pharmacologic activity (enhancement of antigen-specific humoral im-
mune responses and T-cell activation) without any generalized, nonspecific immune-cell acti -
vation was demonstrated in several studies. In addition, there were no hyperplastic,
preneoplastic, or neoplastic lesions in the peripheral blood or lymphoid tissues of any mon-
key, despite long-term treatment at a clearly immunostimulatory dose of 10 mg/kg.
In exploratory studies, only 2 of over 100 monkeys receiving ipilimumab as a single agent
or in combination with various antigens, DNA vaccines, or an anti-CD137 mAb (BMS-
11. Biological therapies for cancer378
III. Specialty biologics and indications
663513), demonstrated adverse toxicities that are considered to be consistent with similar in-
flammatory responses observed in humans (Ryan et al., 1999). These included colitis,
observed in one of six monkeys receiving ipilimumab at 10 mg/kg in combination with three
vaccines (HBsAg, SK-mel, and DNP-Ficoll) in a 3-month exploratory study, and a persistent
dermatitis/rash in the inguinal are a accompanied by peripheral lymphadenopathy that
occurred in one monkey given ipilimumab at 10 mg/kg approximately twice monthly in
combination with another immunomodulatory antibody (a fully human anti-CD137 mono-
clonal antibody) and SIV DNA vaccines in a 4-month pharmacology study. The colitis
occurred about 3 months after study initiation and was characterized by acute to subacute
inflammation, crypt abscesses, and erosion of the colon extending into the rectum. The
dermatitis appeared approximately 4 weeks following the last dose of ipilimumab and was
transiently responsive to antihistamine treatment. The finding was eventually resolved
with prednisone treatment. Skin samples obtained from biopsy of the affected area indicated
mild epidermal thickening/proliferation (increased nuclear proliferation antigen Ki67
þ
) with
the underlying dermis containing mild perivascular edema and infiltrates of lymphocytes
(mostly CD8
þ
), macrophages (CD68
þ
), and mast cells. No infectious agents were present.
The inflammatory responses observed in the monkeys described above are consistent with
the proposed key role of CTLA-4 in maintaining self-tolerance in the immune system and
were similar in nature to the primary adverse events seen with clinical ipilimumab therapy
although they were less frequently observed in monkeys than humans, despite similar expo-
sure to drug and comparable tissue-binding profile. Clinical ipilimumab-associated adverse
events involved predominantly the gastrointestinal tract, skin, liver, endocrine glands, or
CNS (sensory or motor neuropathy) [87]. The most common serious adverse events involved
the lower gastrointestinal tract and clinically manifested as diarrhea. Diarrhea due to treat-
ment with ipilimumab ranges from mild to very severe and may become life-threatening.
The binding of ipilimumab to CTLA-4 expressed on gut-associated lymphoid tissue was
confirmed in human and monkey tissue-binding studies, and suggests that these lympho-
cytes (T cells) exist in an activated state, making them susceptible to CTLA-4 blockade by ipi-
limumab. In the majority of clinical cases, colitis was successfully managed symptomatically
or with short treatment courses of high-dose corticosteroids. Rash is among the most com-
mon adverse event reported and most cases were mild to moderate in severity; pruritus
was also reported (Ryan et al., 1999).
In the monkey toxicity studies, enhancement (2- to 7-fold control across all studies) of
T celledependent antibody responses to antigens (i.e., SK-mel cells, HbsAg, KLH) adminis-
tered in conjunction with ipilimumab was demonstrated. Blockade of CTLA-4/B7 interac-
tions prevents induction of peripheral T-cell tolerance upon vaccination with peptides
under tolerogenic conditions, suggesting that CTLA-4 is actively involved in the induction
of anergy. In addition to being expressed on activated effector T cells, CTLA-4 is constitu-
tively expressed on the surface of T-regulatory cells. T-regulatory cells suppress immune re-
sponses but the role of CTLA-4 in this process is controversial. There were no drug-related
changes in T-regulatory cells (CD4
þ
CD25
þ
, CD4
þ
CD25
þ
CTLA-4
þ
,CD4
þ
CD25
þ
FoxP3
þ
)in
monkeys receiving three monthly doses of ipilimumab at 10 mg/kg.
The observation that CTLA-4 knockout mice suffer a fatal lymphoproliferative disorder sup-
ports the idea that CTLA-4 functions as a key negative regulator of T-cell responses (Chambers
et al., 1997; Tivol et al., 1995; Waterhouse et al., 1995). However, blockade of CTLA-4 function
Bevacuzimab 379
III. Specialty biologics and indications
by ipilimumab did not lead to any generalized multiorgan nonspecific T-cell activation or pro-
liferation, as evidenced by a lack of substantial change in T-cell subsets, nonspecificT-cellacti-
vation, or autoimmune organ pathology in the repeat-dose monkey studies. Further,
antinuclear antibody (ANA) levels were unaffected following an intensive dosing regimen
(four weekly doses of 10 mg/kg) in the 1-month monkey study. In contrast, anti-CTLA-4 anti-
body augmented autoimmune responses in mice prone to specific autoimmune disease (Egen
et al., 2002). Thus, the immune-related toxicities associated with ipilimumab are linked with the
intended pharmacologic basis of CTLA-4 blockade, but were considered to be generally
manageable and considered an acceptable risk for patients with advanced melanoma.
Although there were variable ADCC effects of ipilimumab in vitro, peripheral blood
lymphocyte subset phenotyping analyses performed in the pivotal monkey studies, as well
as the 2-month exploratory study, demonstrated no indication of T-cell depletion (Ryan
et al., 1999). In a 3-month exploratory study, an extended phenotypic analysis demonstrated
an increase (up to 77%, relative to controls), rather than a decrease, in the peripheral blood
CD4 central memory T-cell subset (CD4
þ
CD28
þ
CD95
þ
) in monkeys treated with ipilimumab .
The numbers of Treg cells (CD4
þ
CD25
þ
, CD4
þ
CD25
þ
CTLA-4
þ
,CD4
þ
CD25
þ
FoxP3
þ
), which
express CTLA-4 constitutively, were unaffected in this study.
As with any IV administered protein, ipilimumab has the potential to evoke an infusion
reaction, which may be associated with rapid cytokine release. One monkey in the 4-
month pharmacology study developed an infusion reaction while under ketamine sedation,
accompanied by difficulty in breathing, cyanosis, thready pulse, and muffled heart sounds, at
approximately 5 min following ipilimumab injection on day 58 and approximately 2e3 min
following administration of DNA vaccines that were used to test antigens to assess immune
response. Rechallenge of the monkey with a controlled infusion (3 mL/min) of ipilimumab
approximately 5 months later did not evoke a similar reaction. Since the infusion reaction
was not reproducible after a subsequent controlled ipilimumab rechallenge, the drug rela-
tionship to this event was unclear. However, similar reactions have been documented
following the rapid IV injection of immunoglobulins due to acute cytokine release, and
quickly resolve after decreasing the infusion rate (Misbah and Chapel, 1993). On the day
of the reaction, the infusion rates were not strictly monitored so it is possible that the events
in this particular monkey represented a nonspecific response to rapid immunoglobulin infu-
sion. In addition, the acute reaction could have been further complicated by anesthesia-
induced hypotension during the dose (Misbah and Chapel, 1993; Mertes and Laxenaire,
2002; Yoshizawa et al., 2005; Ochsner, 1977 ). Anesthetics such as ketamine can cause
anaphylactic-like reactions and other abnormalities in animals and humans. The presence
of ADAs is also associated with higher rates of infusions reactions, especially when the pro-
tein therapeutic is administered rapidly (Bendtzen et al., 2006; Mayer and Young, 2006). In
fact, high levels of antiipilimumab antibodies were detected in this monkey prior to the
infusion-related reaction, and could have formed drugeantibody immune complexes upon
rapid infusion of ipilimumab. Immune complex formation and solubility are highly depen-
dent on achieving equivalency of antigen and antibody concentrat ions; thus, by slowing
the infusion rate, the opportunity for soluble immune complexes to form is greatly reduced.
Acute infusion reactions have not occurred in any other monkey toxicology or pharmacology
studies (n ¼ 88 monkeys) conducted with ipilimumab, in which unanaesthetized monkeys
were dos ed and infusion rates were closely controlled.
11. Biological therapies for cancer380
III. Specialty biologics and indications
As with most mAbs, the specificity of ipilimumab and its molecular size limit possible ac-
cess to ion channels in the myocardium, and ipilimumab is not expected to affect ion currents
or channel selectivity as can occur with a variety of small-molecule drugs. Specific safety
pharmacology studies, including cardiovascular telemetry safety pharmacology studies,
were not conducted. In accordance with ICH S7 and S9, evaluations of the potential effects
of IV administration of ipilimum ab on the cardiovascular, central/peripheral nervous, and
respiratory systems were included as part of the pivotal GLP repeat-dose toxicity studies
in monkeys. No drug-related findings were observed in standard clinical evaluations of car-
diovascular, respiratory and neurologic function (Ryan et al., 1999).
The toxicology testing paradigm for ipilimumab investigated the toxicological profile of
the drug substance/product in its entirety (Ryan et al., 1999). All pivotal studies were con-
ducted with product that was comparable with clinical material in use at the time the studies
were conducted based on comprehensive analytical evaluations of drug product and PK
equivalence. The drug substance may have included residual impurities from the
manufacturing process and any unknown impurities that were not detected using standard
detection assays. However, during process validation, experiments confirmed consistent
reduction of impurities throughout the purification process for all impurities inherent in
the processing of ipilimumab, such as DNA, recombinant human insulin, methotrexate,
endotoxin, and host-cell protein. On the basis of these results, it was concluded that potential
process-related impurities were removed to acceptable levels. The presence of potential
degradation products or aggregation products was routinely analyzed at the release of the
drug substance/product. Excipients included in the drug product were of pharmaceutical
grade. Thus, toxicity testing of excipients and impurities was not done. Any toxicities
observed to result from ipilimumab administration during the toxicology program were
the result of the total drug substance/product.
Administration of systemic corticosteroids may be required in clinical studies with ipilimu-
mab to treat immune-related adverse events; thus, it was of interest to determine how con-
current or sequential treatment with dexamethasone affe cts anti-CTLA-4 mAb antitumor
effects. These studies showed that concurrent treatment with dexamethasone reduced the
antitumor activity elicited by anti-CTLA-4 mAb; however, administration of dexamethasone
after multiple anti-CTLA-4 mAb treatments did not negate the antitumor effect elicited by
CTLA-4 blockade. Therefore, these data support the use of corticosteroids for the treatment
of immune-related adverse events, which may occur during anti-CTLA-4 immunotherapy.
In accordance with ICH S9, no standard genotoxicity or carcinogenicity studies were per-
formed with ipilimumab.
Routine histopathological examinati on of organs collected in toxicity studies indicated that
there were no effects on the male and female reproductive organs including sperm and fol-
licle/ovum morphology and maturation. The effects of ipilimumab on reproduction and
development were studied in an enhanced PPND study in cynomolgus monkeys. This study
was submitted as a postmarketing Phase IV commitment. In the PPND study in cynomolgus
monkeys, pregnant monkeys received ipilimumab every 21 days from the beginning of
organogenesis in the first trimester through delivery, at dose levels of 10 mg/kg or 30 mg/
kg. Serum ipilimuma b concentrations in the offspring were similar to those in the mo thers
for up to 3 months postpartum. Ipilimumab was shown to be present at very low levels in
milk from adult mothers (with mean milk: serum ipilimumab concentration ratios that
Bevacuzimab 381
III. Specialty biologics and indications
were 0.002e0.003). Immunogenicity to ipilimumab occurred in 26% of the adult females
during the gestation and/or postpartum periods with sustained positive ADA responses in
five mothers each at 10 and 30 mg/kg; the impact on maternal serum concentrations in these
animals varied from slight to significant. There was no demonstrable relationship between
ADA formation and pregnancy outcome. Positive ADA titers were detected in two infants,
but appear to have been of maternal origin and transferred prenatally since the mothers of
these animals had high ADA titers and the AD As were cleared in manner/timing consistent
with the maternally transferred ipilimumab. The presence of maternal antibodies in these an-
imals did not appear to have any adverse effects on the infants or their ability to mount a
T celledependent antibody response (TDAR) to HBsAg or tetanus toxoid.
There were no adverse ipilimumab-related effects on maternal clinical observations, food
consumption, body weights, clinical pathology, serum Ig or ANA levels, or lymphocyte sub-
populations during gestation through 6 months postpartum. No treatment-related adverse ef-
fects on reproduction were detected during the first two trimesters of pregnancy. Maternal
pregnancy outcomes for the first two trimesters were comparable in control and drug-
treated groups. Beginning in the third trimester (GD100), the ipilimumab groups experienced
increased maternal weight decrements; higher incidences of abortion, stillbirth, premature de-
livery (with corresponding lower birth weight); and higher incidences of infant mortality in a
dose-related manner, compared with controls (21% and 30% for 10 and 30 mg/kg/every
3 weeks, respectively; compared with study controls [0%] and historical controls [17.6%]).
Some infant mortality in ipilimumab-treated groups could be attributed to prematurity; how-
ever, the group mean durations of gestation were comparable in the three experimental groups.
Examination of mothers with thi rd trimester losses and infant losses including pathology
endpoints from the placenta, umbilical cord, or the fetus/infant itself did not reveal a clear
cause for ipilimumab-related death or pregnancy failure. The pathology of the third trimester
fetal losses and early infant mortality was most consistent with a nonspecific failure to thrive.
Considering the third trimester pregnancy losses, shortened gestation length, infant prema-
turity, and infant loss observed in the study combined with the mechanism of action of ipi-
limumab (i.e., inhibition of CTLA4 and immunostimulation) and the localized expression of
the target in the placenta, a drug relationship to the observed developmental mortality could
not be ruled out.
Urogenital malformations were observed in two infants of mothers treated with 30 mg/kg
per dose. Infants exposed to ipilimumab at 30 mg/kg every 3 weeks had a lower mean body
weight at birth. However, the rate of body weight gain in these infants increased by 3 months
of age and mean body weight for the 30 mg/kg/every 3 weeks group was comparable with
that of the control infants at 6 months.
Ipilimumab did not adversely affect the ability of infants to mount a TDAR at six or
18 weeks of age. There were no adverse effects observed in infants related to ipilimumab
exposure in utero with respect to clinical observations, morphometric measurements, neuro-
behavioral and skeletal evaluations, clinical pathology, lympho cyte phenotyping, ANA for-
mation, or serum immunoglobulin levels through 6 months postnatal.
The observation that CTLA-4 knockout mice suffer a fatal lymphoproliferative disorder
supports the idea that CTLA-4 functions as a key negative regulator of T-cell responses (Ton-
nesen et al., 2000; Health Canada, 2004). However, blockade of CTLA-4 function by ipilimu-
mab in pregnant monkeys during gestation did not lead to clinically relevant, generalized
11. Biological therapies for cancer382
III. Specialty biologics and indications
multiorgan autoimmune pathology or changes in T-cell subsets in infants followed out to
6 months of age. The infants were immunocompetent and able to mount a robust TDAR
as early as 6 weeks of age. Furthermore, ANA levels were unaffected in infants of monkeys
receiving an intensive regimen or ip ilimumab (30 mg/kg every 3 weeks) in the PPND study.
Thus, it appears that blockade of CTLA-4 does not afford the same consequences as complete
deletion from the genome at conception (Howdieshell et al., 1998).
In accordance with the ICH S6(R1) guideline and the ICH S1A guideline on the need for
carcinogenicity studies of pharmaceuticals, no studies on genotoxicity or carcinogenicity
were conducted, which was acceptable. In addition, considering the lack of relevance of
the rodent species for ipilimumab, the limited value of short-term carcinogenicity studies
with homologous products, and the advanced condition intended to be treated by ipilimu-
mab, the lack of conventional carcinogenicity studies was accepta ble.
Local tolerance was assessed as part of the toxicity studies. Ipilimumab was administered
from preformulated (ready-to-use) vials at the clinical concentration (generally w5 mg/mL).
However, injection rates were faster and varied from w3e10 mL/min (up to 50 mg/min) in
the nonclinical studies compared with 90-min clinical infusion (total dose of 210 mg for a
70 kg person, up to 2.3 mg/min), thus, providing a safety factor of approximately up to
w22-fold based on infusion rate. No substantial irritation at the injection site was observed.
As a selective immunomodulator, ipilim umab was expected to have effects on the immune
system. The immunologic effects of ipilimumab (including special assessments of biomarkers
of mechanism of action) were studied in the pivotal and exploratory repeat-dose studies in
monkeys. Specifically, in addition to standard immune hematologic (i.e., leukocyte counts
and differentials) and clinical chemistry (i.e., globulins) assessments and gross and histopath-
ologic examinations of lymphoid tissues included in the repeat-dose studies, specialized im-
mune parameters were incorporated into several of the studies including peripheral blood
lymphocyte phenotyping (including activated T-cell and regulatory T-cell subsets), lympho-
cyte phenotyping in spleen, inguinal lymph node, and colon epithelium, ANA assessments,
TDAR assessments, delayed-type hypersensitivity assessments, and intracellular staining of
ex vivo stimulated cytokine (IL-2, TNFa and/or IFNg) production by monkey peripheral
blood T cells. Ipilimumab increased antigen-specific humoral and cellular immune responses
in monkeys, but did not cause global nonspecific immunostimulatory or aut oimmune toxic-
ities in the majority of animals (except the two cases already mentioned).
In conclusion, the most notable concern identified during nonclinical testing was a low
incidence of serious immune-related adverse events, manifested as colitis or dermatitis, in
two out of over 100 monkeys dosed with ipilimumab (Ryan et al., 1999). These inflammatory
responses are consistent with the prop osed key role of CTLA-4 in maintaining self-tolerance
in the immune system and subsequent immunostimulation afforded by blockade of this re-
ceptor and are similar in nature to the primary adverse events seen with clinical ipilimumab
therapy. It is assumed that these observations should be taken as a general indication that
ipilimumab might be associated with immune-related serious adverse effects, which cannot
always be predicted in character from animal studies. Ipilimumab was also a selective devel-
opmental toxicant when administered to pregnant monkeys during the period of organogen-
esis through parturition at doses of 10 and 30 mg/kg once every 3 weeks and is not
recommended for use during pregnancy unless the potential benefit justifies the potential
risk to the fetus (Howdieshell et al., 1998).
Bevacuzimab 383
III. Specialty biologics and indications
PD-1 blockade and DART
Nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), durvalumab
(Imfinzi), and avelumab (Bavencio), all target the programmed cell death-1/programmed cell
death Ligand 1 (PD-1/PD-L1) pathway. Among these, nivolumab and pembrolizumab are
two of the most well described checkpoint inhibitors in immuno-oncology. Instead of review-
ing the nonclinical programs, which are described in detail elsewhere (Fu et al., 2017; Ham-
mond et al., 2018; Hutchins et al., 2020; Kumar et al., 2021), a discussion of a unique aspect of
their nonclinical development, the DART strategy, is discussed here.
PD-1 is a protein receptor that is expressed on the surface of T cells and other immune
cells, including B cells, natural killer cells, macrophages, dendritic cells, and monocytes
(Han et al., 2020). Its main function is to regulate the immune response and prevent the im-
mune system from attacking healthy cells and tissues in the body. When PD-1 interacts with
its ligands, PD-L1 or PD-L2, which are expressed on the surface of certain cells such as tumor
cells and infected cells, it sends a negative or tolerogenic signal to the T cell, preventing auto-
immunity and excessive immune responses that can damage healthy tissues. Cancer cells and
viruses can exploit this mechanism to evade immune attack by upregulating the expression of
PD-L1 on their surface, which allows them to escape immune recognition and destruction by
T cells. Drugs that block the interaction between PD-1 and PD-L1, therefore, restore the ability
of T cells to recognize and destroy cancer targets and have shown promising results against
multiple tumor types in clinical trials.
The PD-1/PDL-1 pathway’s tolerogenic effect has also been found to play a role in preg-
nancy, specifically, in maintaining the tolerance of the mother’s immune system to the devel-
oping fetus (Guleria et al., 2005; Prell et al., 2016; Li et al., 2021a,b). During pregnancy, the
fetus expresses paternal antigens, which can be recognized as foreign to the maternal immune
system. Erroneous immune responses to fetal antigens could potentially lead to miscarriage,
preeclampsia, or prete rm labor (Zhang et al., 2018). The presence of PD-L1 on numerous tis-
sues associated with the developing fetus acts as a barrier to maternal antife tal immune re-
sponses, promoting a Treg-driven tolerogenic environment. Studies have shown that
blocking PD-1 or its ligands in animal models of pregnancy can lead to fetal rejection and
miscarriage. Therefore, PD-1 is believed to play an essential role in maintaining immune
tolerance toward the developing fetus during pregnancy. Based on the biology and expres-
sion profile of PD-1/PD-L1 it was theorized that blockade of that pathway may lead to dys-
regulation of the immune response at the maternalefetal interface.
Multiple PD-1/PD-L1 pathway blockers have not conducted dedicated DART studies as
part of their nonclinical toxicology programs, instead opting to perform literature-based as-
sessments of the target expression pattern, kinetics of expression, and allogenic mouse preg-
nancy models utilizing PD-1 deficient mice (Poulet et al., 2016; FDA Atezo Pharmacology
Review; EMA Assessment Report:Atezolizumab). Collectively, the available nonclinical
studies and literature review support ed a position that PD-1/PD-L1 blockade leads to a
loss of fetal tolerance and an increased risk of immune-mediated abortion. The literature-
based assessments were considered sufficient to inform potential risks to human pregnancy
for marketing authorization without demonstration of product-specific DART risks. Nivolu-
mab and durvalumab are exceptions to the literature -based approach; in vivo NHP ePPND
studies directly demonstrated an increased rate of abortion, premature delivery and
11. Biological therapies for cancer384
III. Specialty biologics and indications
premature infant death was noted with administration (Opdivo Prescribing Information,
Imfinzi Prescribing Information). However, the prescribing information for all of the PD-1/
PD-L1 blockers listed above carry similar warnings of potential fetal harm when adminis-
tered to pregnant women, and advises the use of effective contraception during treatment
and for several months after the last dose (Tecentriq: Highlights of Prescribing Information
,
Keytruda Prescribing Information, Opdivo Prescribing Information Imfinzi Prescribing Infor-
mation, Bavencio Prescribing Information).
Given the recent guidance from FDA on the use of NHPs for DART studies (discussed
above) early outreach to Regulatory Agencies around the use of literature-based DART as-
sessments versus in vivo NHP DART studies is strongly recommended.
Development of relatlimab as a combination immunotherapy
Relatlimab (anti-LAG-3; BMS-986016) is a human IgG4 antibody that binds LAG-3 with
high affinity and blocks its interaction with ligands MHC II and fibrinogen-like protein-1
(Thudium et al., 2022). LAG-3 is a new generation immune checkpoint inhibitor molecule
that is upregulated on the surface of T cells and inhibits T cell activity in a manner indepen-
dent from that of PD-1 and CTLA-4. LAG-3 and PD-1 have been reported to have extensive
coexpression on tumor-infiltrating lymphocytes (Woo et al., 2012; Gros et al., 2014). The
expression profile along with proof-of-concept pharmacology data strongly supported relat-
limab as an attractive and rational combination partner for nivolumab in the clinic (Andrews
et al., 2017). Following Phase 1/2 and Phase 2/3 trials of relatlimab in combination with nivo-
lumab, the combination immunotherapy was approved by the FDA and EMA in 2022 for the
treatment of unresectable and metastatic melanoma. In the pivotal trial, the combination
immunotherapy more than doubled the median progression-free survival compared with
nivolumab monotherapy (Chocorro et al., 2022; Tawbi et al., 2022). This fixed-dose combina-
tion is marketed under the name Opdualag (nivolumab and relatlimab-rmbw). Consistent
with the heightened potential for autoimmunity with combination immune checkpoint inhi-
bition, prominent warnings for Opdualag include immune-related adverse reactions
including pneumonitis, colitis, hepatitis, endocrinopathies, dermatologic reactions, nephritis,
and myocarditis (Opdualag: Highlights of Prescribing Information).
Genetic ablation of both LAG-3 and PD-1 (Lag3
/
Pdcd1
/
) in mice results in autoim-
mune disease manifestation and pathology as they age. Lag3
/
Pdcd1
/
mice developed
an early onset (w4 weeks of age) lethal autoimmune condition that resulted in w80% of
the mice moribund by w10 weeks with major histopathological manifestations of fibrosing
lymphohistiocytic endocarditis, myocarditis and pancreatitis (Woo et al., 2012). LAG-3 and
PD-1 single knockout mice, by contrast, lacked disease manifestations or histopathology
over the same period of observation. Although Lag3
/
Pdcd1
/
mice present with autoim-
munity, they demonstrated better tumor growth control and survival over single knockout or
wildtype controls. Pharmacological inhibition of LAG-3 and PD-1 with surrogate antibodies
also resulted in synergistic antitumor efficacy against established syngeneic tumors in wild-
type mice, which were otherwise largely resistant to single agent treatment (Woo et al., 2012;
Thudium et al., 2022). These data provided strong proof-of-concept for synergistic coopera-
tion of LAG-3 and PD-1 in limiting tumor growth.
Bevacuzimab 385
III. Specialty biologics and indications
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