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Monoclonal antibodies
Monoclonal antibodies developed for oncology have various mechanisms of action, which
must be taken into account when designing a nonclinical development package. They can
mark the cancer cell to make it more visible to the immune system by attaching to specific
proteins found on B cells or T cells (i.e., CD20, CD52 antigens) (Schuster et al., 2006). For lym-
phomas, the immune cells themselves are the cancer and targeting those is the desired mech-
anism of action (i.e., rituximab). Rituximab binds to the CD20 antigen on B cells (and B-cell
lymphomas) and causes a depletion of these cells through an antibody-mediated cell cytotox-
icity mechanism. MAbs that react with specific types of cancer may enhance a patient’s im-
mune response to the cancer and other mAbs may actually target or signal immune cells to
become or remain activated to attack cancer cells within the body. Ipilimumab, an antibody
against the costimulatory CTLA-4 receptor, is one such example. The proposed mechanism of
action for ipilimumab is interference of the interaction of CTLA-4, expressed on a subset of
activated T cells, with B7 (CD80/CD86) molecules on professional antigen-presenting cells.
This results in T-cell potentiation due to blockade of the inhibitory modulation of T-cell acti-
vation promoted by the CTLA-4/B7 interaction. MAbs can also block growth factor receptors
and inhibit the growth of new cells. Epidermal growth factor receptor (EGFR) is one receptor
that accepts growth signals and is expressed on the cell surface of normal cells and cancer
TABLE 11.2 Select list of marketed biologics for oncology indications.dcont’d
Trade name Generic name Target/marker Other characteristics
Ontak Denileukin
diflitox
Recombinant DNA-derived
cytotoxic protein composed
of the amino acid sequences
for diphtheria toxin
fragments A and B (Met1-
Thr387)-His and the
sequences for human IL-2
(Ala1-Thr133)
Proleukin Aldesleukin Recombinant IL-2 (des-
alanyl-1, serine-125 human
IL-2)
Differs from native IL-2 in that it is not
glycosylated, it has no N-terminal alanine, the
molecule has serine substituted for cysteine at
amino acid position 125, and the aggregation
state of proleukin is likely to be different from
that of native IL-2
Roferon Interferon
alpha-2a
Recombinant interferon
alpha 2a
Nonspecific immunomodulators
Theracys,
TICE BCG
BCG Attenuated, live culture
preparation of the bacillus
CalmetteeGuerin (BCG)
strain of Mycobacterium bovis
G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; KGF, keratinocyte
growth factor.
11. Biological therapies for cancer366
III. Specialty biologics and indications
cells. Human epidermal growth factor receptor (HER) binds to cell surface receptors and reg-
ulates normal cell proliferation and survival. There are four known HER proteins: HER1, -2,
-3, and -4. Vascular endothelial growth factor (VEGF) inhibits the ability of tumors to form
new blood vessels to feed their growth (Schuster et al., 2006). When a radioactive particle
is combined with an mAb, radiation is delivered directly to cancer cells. Radiation-linked
mAbs deliver a low level of radiation over a longer period of time and can be as effective
as high-dose external beam radiation (Gregory et al., 2009). For example, the mAb ibritumo-
mab attaches to receptors on cancerous blood cells to deliver radiation. Antibodies carrying
radioisotopes may also prove useful in diagnosing certain cancers, such as colorectal, ovarian,
and prostate. Chemotherapeutic agents, toxins, or other molecules also can be attached to
mAbs. The attached molecules are inactive until they have reache d the target cell, where
they are activated, causing cell death and minimizing harm to healthy cells (Sharkey and
Goldenberg, 2006). In the case of con jugated molecules, the chemical entity that is attached
to the biologic may be subject to a toxicology evaluation typical for small-molecule drugs
wherein a second species (i.e., a rodent) may, for example, be used to characterize toxicity
of that specific portion of the drug.
Rituximab
Rituximab (Rituxan, MabThera) is a chimeric mouse/human monoclonal antibody, that
targets the CD20 antigen on human B lymphocytes with high specificity. Rituximab is
approved for use in non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, rheumatoid
arthritis, Wegener’s granulomatosis, and microscopic polyangiitis (Coiffier, 2007; Kimby,
2005). The major adverse effects of rituximab in humans are acute allergic and infusion reac-
tions occurring in 25% of oncology patients (Kimby, 2005; Fleischmann, 2009). Boxed warn-
ings include fatal infusion reactions, tumor lysis syndrome, severe mucocutaneous reactions,
and progressive multifocal leukoencephalopathy (PML) (EMA, 2005). Anti-CD20 mAbs are
also associated with late-onset neutr openia, slow recovery of normal B cells, infections, reac-
tivation of hepatitis, intestinal perforation, and interstitial pneumonia (Ram et al., 2009).
The toxicology development package was performed in compliance with ICH regulations,
utilized the same formulation used clinically and the molecule was ultimately marketed
(EMA, 2005). Since rituximab did not bind to rodent or canine B cells, the nonclinical devel-
opment was conducted in NHPs (Reff et al., 1994; Goldenberg et al., 2010; Gaufin et al., 2009).
Rituximab was administered intravenously and was well tolerated in general toxicology
studies (single-dose toxicity studies in cynomolgus monkeys at doses up to 100 mg/kg
and multiple-dose toxicity studies at doses up to 20 mg/kg/week) (Bugelski and Martin,
2012). Because of the high specificity and lack of off-target binding of rituximab, no toxic ef-
fects other than the expected pharmacological depletion of B cells in peripheral blood as well
as in lymphatic tissues was observed in toxicity studies. The B-cell population showed recon-
stitution after cessation of treatment. Apart from the expected depletion of B cells, there were
no other findings in clinical pathology and histopathology. In particular, there was no
evidence of toxicity to the hematopoietic system, T cells or other cells of the non-CD20
þ
lymphocyte lineage. Rituximab does not affect the functionality of the pool of CD20-
antibodyeproducing plasma cells. Although none of the rituximab-treated monkeys
Rituximab 367
III. Specialty biologics and indications
developed any signs of infections, specific experiments to demonstrate the difference between
overall and B cellespecific immunosuppression were performed in preclinical studies.
Although the risk for developing an immune or allergenic response following treatment
with rituximab is also very low, since rituximab does not deplete antibody-producing plasma
cells, patients with an existing allergy to murine proteins have been advised to avoid treat-
ment with rituximab. In several studies, the induction of antibodies (monkey antichimeric an-
tibodies or antidrug antibodies (ADAs)) against rituximab was reported and could have
rendered some of the results of toxicity studies inconclusive. This is likely due to the sequence
differences between the monkey IgG and both parts of the chimeric antibody. However, the
immunogenicity of rituximab in preclinical studies had no predictive value for immunoge-
nicity in the clinic. The longest duration toxicity study included in the initial marketing appli-
cation was 3 months and the lack of chronic toxicity studies was labeled a “clear-cut
deficiency” by some health authorities at that time (prior to the adoption of the ICH S9 guid-
ance), but additional animal studies were not indicated given the amount of existing clinical
data. In keeping with this precedent, the current ICH S9 guidance advises that, for most anti-
cancer pharmaceuticals, nonclinical studies of 3 months duration would be considered suffi-
cient to support marketing (ICH S9, 2010).
Pharmacodynamic studies investigated distribution to immune organs and immunopath-
ologic analysis of rituximab. A repeat-dose pilot pharmacology/toxicology study where cyn-
omolgus monkeys were administered a dose of 16.8 mg/kg demonstrated the relationship
between repeated exposure to rituximab and B-cell depletion in the peripheral blood, bone
marrow, and lymphatic tissues. This experiment formed the basis for the initial dose-
escalating study in humans. In a 2-month study in monkeys, weekly doses of rituximab up
to 20 mg/kg were generally well tolerated with occasional emesis. Findings of testicular
hypospermatogenesis or aspermatogenesis and thymic lymphoid atrophy in monkeys were
observed. However, since rituximab had no direct binding specificity or cytotoxicity to testic-
ular or thymic tissues in cross-reactivity studies with human tissues the testicular findings
were attributed to sexual immaturity of the monkeys and thymic atrophy was attributed
to experimental stress. Although ICH S9 was not in effect at the time, ICH guidance relating
to oncology drugs and biotherapeutics were followed and as a result, no studies to assess
mutagenicity, genotoxicity, or carcinogenicity were performed to support the initial market-
ing application of rituximab. Reproductive studies were not completed prior to marketing;
however, these studies were completed during Phase IV (postmarketing).
In EFD and PPND toxicity studies, female cynomolgus monkeys were administered ritux-
imab from gestation day (GD) 20e50 for the EFD study and GD20 to postpartum day 28 for
the PPND study (Bugelski and Martin, 2012; Vaidyanathan et al., 2011). Placental transfer of
rituximab was demonstrated at GD100 and fetuses demonstrated B-cell depletion in
lymphoid tissues at maternal exposures similar to human therapeutic exposures (Rituxan
USPI, 2014). There was no significant effect on T celledependent antibody responses
following vaccination or antigenic challenge, and recovery of B cells in the infants was
demonstrated.
The mechanism underlying B-cell depletion and potential cell-mediated B-cell killing in
monkeys has not been fully elucidated nor has the question of whether rituximab is internal-
ized or not after binding to CD20 on human lymphocytes (Coiffier, 2007). Some of the short-
comings of animal safety data are now complemented by clinical data from patients with
11. Biological therapies for cancer368
III. Specialty biologics and indications
malignant lymphoma, showing that plasma concentrations are inversely correlated with fre-
quency of adverse events (i.e., more CD20-binding sites and lower plasma concentrations are
more likely to induce flu-like syndr ome).
Bevacuzimab
Bevacizumab (Avastin) is a humanized recombinant IgG1 version of the murine antihuman
VEGF monoclonal antibody A4.6.1 (muMAb VEGF) (EMA, 2005; Kim et al., 1992). Bevacizu-
mab consists of about 93% human and 7% murine protein sequence, with the murine se-
quences comprising the complementarity-determinant region of the molecule. VEGF is a
major regulator of angiogenesis during normal and pathological processes, including those
associated with tumor growth (reviewed in Ferrara and Gerber (2001) and Ferrara and
Davis-Smyth (1997)) and has a key regulatory function during developmental angiogenesis
(Ferrara et al., 1996; Carmeliet et al., 1996). Bevacizumab selectively binds with high affinity
to all isoforms of human VEGF and neutralizes its biologic activity through a steric blocking
of interaction with receptors, VEGFR-1 and VEGFR-2, on the surface of endothelial cells
(EMA, 2005). Since VEGF receptors are significantly upregulated in the tumor vasculature,
the neutralization of VEGF by bevacizumab provides the rationale for a relatively selective in-
hibition of the tumor angiogenesis and, thereby, inhibition of tumor growth and metastasis.
The pharmacological activities of bevacizumab were evaluated in a number of in vitro as-
says using recombinant human VEGF. Bevacizumab and the parent murine antibody A4.6.1
were compared for their ability to inhibit bovine capillary endothelial cell proliferation in
response to VEGF. The two mAbs were essentially equivalent, both in potency and efficacy
when tested with human cells, human tissues or human VEGF isoforms (Kim et al., 1992; Pre-
sta et al., 1997). In vivo, the pharmacology of an anti-VEGF approach was investigated pri-
marily in murin e xenograft models using A4.6.1 and resulted in marked growth inhibition
of a variety of tumor types, including rhabdomyosarcoma (Kim et al., 1993; Borgstrom
et al., 1996), glioblastoma (Yuan et al., 1996; Gerber et al., 1998), leiomyosarcoma (Yuan
et al., 1996), ovarian carcinoma (Mesiano et al., 1998), prostate carcinoma (Borgstrom et al.,
1998), colon adenocarcinoma (Warren et al., 1995), Wilms’ tumor (Soffer et al., 2001), hepato-
blastoma (McCrudden et al., 2003), neuroblastoma (Dvorak et al., 1995), breast carcinoma
(Sweeney et al., 2001), melanoma (Yuan et al., 1996), and pancreatic cancer (Hotz et al.,
2003). The density of vessels was also decreased in the antibody-treated tumors (Yuan
et al., 1996). In three different animal models inoculated with human tumor cells (colon car-
cinoma, prostate and Wilms’ tumor), treatment with A4.6.1 markedly inhibited primary tu-
mor growth and reduced metastasis dissemination to liver and lung (Ku et al., 1993).
Several nonclinical studies were conducted in order to study the pharmacologic effect of
anti-VEGF antibody treatment in combination with other antineoplastic treatments in animal
models inoculated with cells from human tumor cell lines. Combination of A4.6.1 or bevaci-
zumab with radiation, paclitaxel, doxorubicin, topotecan, or docetaxel often resulted in addi-
tional tumor growth inhibition compared with monotherapy (Clauss et al., 1990; Lee et al.,
2000; Hu et al., 2002; Kim et al., 2002).
Initial toxicology studies were performed in cynomolgus monkeys and rabbits using an
intravenous route of administration as is used clinically. At the time of nonclinical
Bevacuzimab 369
III. Specialty biologics and indications
development, cynomolgus monkey VEGF was predicted to have a protein sequence identical
to that of human VEGF (Shima et al., 1996) and bound to bevacizumab with affinity similar to
human VEGF. Bevacizumab was also found to bind rabbit VEGF, although with a lower af-
finity than for human and cynomolgus VEGF; the dissociation constant (K
d
) is 8.0 nM for rab-
bit VEGF compared with 1.1 nM for human VEGF. Several non-GLP investigative
experiments in rabbits were designed to explore findings observed in human clinical trials
or investigate known toxicities or risks (perturbation of growth plate morphology and
ovarian function, wound-healing, coagulation and renal function studies) (Goldenberg
et al., 2010; Gaufin et al., 2009). Due to the lower affinity of bevacizumab to rabbit VEGF
compared with human VEGF, the studies in rabbits used higher doses and were of shorter
duration to avoid the development of ADAs. All toxicology studies with bevacizumab
were conducted according to GLP, with the exception of the investigational/specialized
studies to further investigate known toxicities or risks (Fleischmann, 2009). No single-dose
toxicity studies were performed and no stand-alone in vivo safety pharmacology studies
were conducted with bevacizumab or A4.6.1. Safety pharmacology endpoints were evaluated
as part of the toxicology studies. The pivotal toxicity studies were conducted only in mon-
keys, as bevacizumab was generally not immunogenic in this species, and entailed bevac izu-
mab administration alone or in combination with commonly used chemotherapy regimens
for up to 26 weeks.
Repeat-dose IV studies of 4 or 13 weeks duration were conducted in which young adult to
adult cynomolgus monkeys were administered 2, 10, or 50 mg/kg twice weekly or 2, 10, or
50 mg/kg weekly, respectively ( Ryan et al., 1999), and a 26-week IV study with a 12-week
recovery where animals were administered 2, 10, or 50 mg/kg once weekly or 10 mg/kg
twice weekly was conducted in sexually mature (4- to 7-year-old) adult cynomolgus monkeys
(Fleischmann, 2009). Bevacizumab was generally well tolerated at doses up to 50 mg/kg.
Treatment-related effects were consistent with the inhibition of VEGF-dependent angiogen-
esis. The primary drug-relate d effects of bevacizumab involved sites of active neo-
angiogenesis and included an increase in physeal dysplasia characterized by thickening of
the growth plate, clusters of hyperplastic/hypertrophied chondrocytes, subchondral bony
plate formation, and inhibition of vascular invasion of the long bone growth plates with a
distinct zone of cessatio n of bone growth and occ asional linear fissuring in young adult cyn-
omolgus monkeys (Warren et al., 1995). Physeal dysplasia occurred in males at 2 mg/kg and
in females at 10 mg/kg in the 13-week study and was observed at slightly higher doses in the
4-week study. Physeal dysplasia occurred only in actively growing animals and tended to be
reversible. In the 26-week study, the severity of physeal dysplasia was less pronounced than
in the previous studies, likely because an effort was made to select animals with closed
growth plates by performing radiography prestudy. Based on weekly dosing in the 4- and
13-week studies, this effect occurred at exposure levels approximately 1.5- to 2.0-fold below
the expected human clinical expo sure, based on average serum concentrations. In addition, in
the 4-week study enlarged spleens were reported for one male and one female in the 50 mg/
kg group and one male given 50 mg/kg had slight, multifocal renal hemorrhage without cor-
responding pathologic changes to explain the finding. Antibodies to bevacizumab were not
detected in any animal at any time point (Fleischmann, 2009).
In the 13-week study, ovarian and uterine weights were reduced in females at 10 or
50 mg/kg coinciding with a reduced number or absence of corpora lutea. This change was
11. Biological therapies for cancer370
III. Specialty biologics and indications
partially reversible following a 4-week recovery period. In the 26-week study, mildly lower
albumin and albumin-to-globulin ratio and moderately higher glob ulin levels were observed
in males at 10 mg/kg (twice weekly) and 50 mg/kg. In addition, reversible decreases in body
weights, body weight gain, and food consumption occurred in males in these groups. It is
possible that the animals may have grown at a slower rate because of physeal dysplasia.
In females, a dose of 10 mg/kg or higher reduced endometrial proliferation, produced lower
uterine weights and decreased the incidence of menstrual cycles. At 50 mg/kg and 10 mg/kg
(twice weekly), follicular maturation was inhibited at the early Graafian follicle stage in
ovaries. These effects were reversed following recovery (Fleischmann, 2009).
Given the physeal dysplasia observed in studies of bevacizumab in cynomolgus monkeys
following 4e26 weeks of treatment, an investigative study in rabbits was conducted to assess
the suitability of the rabbit for further study of physeal dysplasia. In contrast to the effect
noted in monkeys, bevacizumab did not inhibit vascular invasion or induce subchondral
bony plate formation in rabbits at doses up to 75 mg/kg. As anticipated, antibevacizumab
antibodies were detected in rabbits after multiple dosing. The time course of antibody devel-
opment was thus studied in the rabbit so that further studies conducted could be optimized
to avoid antibevacizumab antibodies. In one study in rabbits ( N ¼ 4), bevacizumab vehicle or
10 mg/kg of bevacizumab were administered intravenously on days 1, 4, 8, and 11. Anti-
bodies developed between 8 and 11 days after the initiation of dosing. Therefore, the duration
of dosing in rabbits was limited by development of antibodies to bevacizumab and this short
exposure period may possibly be insufficient for physeal dysplasia to develop (see Other
Toxicity Studies, below) (Fleischmann, 2009).
New Zealand White (NZW) rabbits were also used to further investigate the effect of bev-
acizumab on luteal function. Rabbits were given three to four IV injections of bevacizumab at
2, 10, or 50 mg/kg or bevacizumab vehicle over a 9- to 10-day period. Bevacizumab exposure
inhibited the function of the corpus luteum at a dose of 50 mg/kg given every 2e3 days in
the rabbit, confirming the finding observed in cynomolgus monkeys. No significant effects
were observed at 10 mg/kg.
The toxicity of bevacizumab in combination with chemotherapeutic regimens (IFL (irino-
tecan/5-fluorouracil/folinic acid), or cisplatin plus paclitaxel) was investigated in 2-week
GLP-compliant monkey studies (Fleischmann, 2009). Administration of IFL resulted in diar-
rhea, body weight loss, and decreased food consumption, in addition to a decreased white
blood cell count and, in some of the animals, a small thymus with corresponding lymphoid
depletion. Microscopic effects noted in the sternal bone marrow of most animals consisted
mainly of erythroid and/or myeloid hyperplasia. The coadministration of bevacizumab
with IFL did not alter the magnitude of the effects related to treatment with the antineoplastic
therapy regimen. Again, given the advanced oncology indication and that bevacizumab is a
monoclonal antibody, no genotoxicity or carcinogenicity studies were performed (ICH
S6(R1), 2011 ).
Studies of EFD were performed in NZW rabbits (Fleischmann, 2009). Following a pilot
study to determine dose and concentration, a pivotal study was performed where presumed
pregnant rabbits (20/group, with four rabbits per group included exclusively for PK sam-
pling) were treated IV with vehicle or bevacizumab at dose levels of 0 (placebo), 10, 30, or
100 mg/kg on GDs 6, 9, 12, 15, and 18. A dose-related significant decrease in maternal
body weight gain and significant mean body weight loss were observed in the two higher
Bevacuzimab 371
III. Specialty biologics and indications
dose groups. Fetal body weights were significantly reduced in all treatment groups. The
number of late resorptions was increased in the 100 mg/kg dose group, resulting in an in-
crease in the total number of resorptions and the percent dead or resorbed fetuses per litter.
There was a dose-related increase in fetal malformations, and in the two higher dose groups
the number of litters with malformations was statistically significantly increased as compared
with the control group. The most frequent malformations included meningocele, thin skin
over the fontanelle, abdominal distention, rotated hindlimbs, enlarged fontanelle, hypoplastic
ribs, bent ribs, malformed maxilla, irregular tibia, absence of digits, hemivertebrae (cervical,
thoracic and lumbar), and several blood vessel variations. The number of litters with any
alteration in the control, 10, 30, and 100 mg/kg groups was 8 (42%), 9 (53%), 13 (76.5%),
and 19 (95%), respectively. In addition, the number of ossification sites per fetus per litter
for metacarpals was reduced or significantly reduced in all treatment groups. Ossification
site averages were also significantly reduced in the high-dose group for caudal vertebrae
and fore- and hindlimb phalanges. Based on these findings, the maternal NOAEL was
10 mg/kg. The fetal NOAEL was less than 10 mg/kg. Antibevacizumab antibodies were
detected in 12% of treated does plus one animal in the control group, 13% of fetuses from
treated does, and in amniotic fluid from 10% of pregnant does treated with bevacizumab.
Following the administration of a single-dose or multiple doses on GDs 6, 9, 12, 15, and 18
of bevacizumab, drug concentrations in maternal serum and amniotic fluid increased linearly
with dose, while drug concentrations in fetal serum increased less than proportionally. Drug
concentrations were higher in fetal than in maternal seru m (ratio >1) at GD29 following five
doses of bevacizumab, while concen trations were lower in fetal then in maternal serum
(ratio <1) at GD21 following a single dose on GD18 (Fleischmann, 2009).
No PPND studies, including maternal function studies or juvenile studies were performed
and no studies of local tolerance were performed as local tolerance was evaluated in the
repeat-dose toxicity studies.
An in vitro study was conducted with bevacizumab and its vehicle to assess their hemo-
lytic potential and the compatibility with cynomolgus monkey and human whole blood,
serum, and plasma. RhuMAb VEGF (at 5 mg/mL) and rhuMAb VEGF vehicle did not cause
hemolysis of cynomolgus monkey or human erythrocytes and are compatible with cynomol-
gus monkey and human serum and plasma. The tissue specificity of bevacizumab against a
panel of 9 normal rabbit tissues, 30 normal cynomolgus tissues, and 36 normal hum an tissues
was determined via an immunohistochemical technique using 10 and 400 mg/mL of bevaci-
zumab conjugated to biotin. No cross-reactivity between bevacizumab and different tissues
was observed.
A number of specialized investigative studies were conducted with bevacizumab to
further characterize the toxicity observed in repeat-dose toxicity studies and also to study
risks associated with VEGF inhibition that could not be discerned from general toxicity
studies (Fleischmann, 2009). Because of the concern that bevacizumab may delay wound-
healing in patients undergoing biopsy procedures or surgery, the effect of bevacizumab on
wound-healing was investigated using a linear incision model (to mimic a surgical incision)
in rabbits (Howdieshell et al., 1998; Nissen et al., 1998; Tonnesen et al., 2000). Bevacizumab
administration resulted in a significant dose-dependent decrease in the tensile strength of the
wounds in these studies, indicating that bevacizumab interferes with wound-healing in the
rabbit. A second model of wound-healing, the circular wound model (to mimic an ulcerative
11. Biological therapies for cancer372
III. Specialty biologics and indications
lesion), was used in rabbits to confirm the results of the studies using the linear incision
model. Male NZW rabbits were treated with IV injections of bevacizumab (50 mg/kg),
vehicle, or methylprednisolone (35 mg/kg) as a positive control. Injections were given every
other day for 2 weeks. By day 12, the wounds in the bevacizumab vehicle-treated group were
78% closed, while in the methylprednisolone- and bevacizumab-treated groups the wounds
were 33% and 46% closed, respectively. Microscopically, 67% of the wounds had completely
healed in the bevacizumab vehicle-treated group, whereas 50% and 17% of the wounds in
methylprednisolone- and bevacizumab-treated animals, respectively, had undergone com-
plete re-epithelialization. The doseeresponse of this effect was explored in a subseque nt
study. Delays in wound-healing were noted at doses as low as 2 mg/kg given twice weekly
for 2 weeks in the rabbit, with a trend toward a doseeresponse relationship, and evidence of
reversibility upon cessation of treatment. Similar experi ments using the linear incision wound
model were also performed in cynomolgus monkeys. One to three male monkeys per group
were treated with IV doses of bevac izumab (0.5 or 2.0 mg/kg) every other day over 8 days
(total of four doses). The effects of bevacizumab on wound-healing in the monkey were
extremely variable, and no doseeresponse relationship was evident.
To investigate the potential for bevacizumab treatment to increase the incidence of throm-
bosis, an acute rabbit model of thrombosis was used. Male NZW rabbits were treated with
either bevacizumab (75 mg/kg) or bevacizumab vehicle daily for 8 days. Following the
eighth dose, a thrombus was induced. No changes in hematology or coagulation parameters
were noted for either treatment group. No differences were observed between groups in time
to clot formation, clot weight, or cuticle bleeding time (Fleischmann, 2009).
To investigate the bevacizumab-related renal adverse effects noted in the clinic, a prelim-
inary study was conducted to examine the deposition of bevacizumab in the kidney. Two fe-
male NZW rabbits per treatment group were given IV dose s of bevacizumab at 2, 10, or
100 mg/kg or bevacizumab vehicle on days 1 and 3. No changes in clinical signs or body
weight as a result of bevacizumab administration were noted on day 5. Examination of the
kidneys by light and electron microscopy indicated no histological differences between con-
trol and treated animals and no selective deposition of bevacizumab (EMA, 2005).
To evaluate further the functional effects of bevacizumab on the kidneys, an animal model
of cisplatin-induced renal dysfunction was used to simulate a subject with preexisting renal
disease. Seven male NZW rabbits were assigned to each of four groups (28 animals total)
and treated with IV administration of cisplatin (1 mg/kg) or saline alone or concurrent with
bevacizumab (50 mg/kg) or bevacizumab vehicle, respectively. Cisplatin or saline was admin-
istered every other day for 2 weeks (six doses total), and bevacizumab or bevacizumab vehicle
was given at the same time as cisplatin or saline every other day during the second week of
treatment (three doses total). The administration of cisplatin alone or the combination of
cisplatin and bevacizumab resulted in significant decreases in body weight compared with
the control group. Treatment with cisplatin and cisplatin plus bevacizumab resulted in a sig-
nificant increase in serum blood urea nitrogen (BUN) and creatinine at the end of the treatment
period compared with the levels in the control group and the group treated with bevacizumab
alone. Treatment with bevacizumab alone did not affect BUN or creatinine. No effects of bev-
acizumab treatment were noted on urinary specific gravity (Fleischmann, 2009).
Bevacizumab treatment alone induced no alteration in any of the endpoints studied in this
model. Two studies of protein (bovine serum albumin, BSA) overload in the rabbit were used
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III. Specialty biologics and indications
to complement the cisplatin model to further examine the effect of bevacizumab on renal
function in the presence of different types of preexi sting renal damage (Fleischmann, 2009).
Rabbits were treated with BSA (5 or 20 mg/kg) for 6 weeks followed by four doses of beva-
cizumab at 50 mg/kg during weeks 6 or 8. No significant differences in serum BUN or creat-
inine were observed following bevacizumab administration.
The interaction potential of bevacizumab and chemotherapeutic agents was studied in two
studies in cynomolgus monkeys. No PK interactions between bevacizumab and cisplatin,
paclitaxel were observed (data not shown). Irinotecan and 5-FU disposition were unchanged
by the concomitant adm inistration of bevacizumab (Fleischmann, 2009).
In summary, based on the panel of nonclinical studies performed, the concerns for use of
bevacizumab in humans are the risk of impaired bone growth in growing individuals, the
risk of impaired wound-healing, and adverse effects on fertility and fetal development.
Trastuzumab
Trastuzumab (Herceptin, anti-p185, rhuMab HER2) is a humanized mAb that binds to the
HER2 protein [81]. HER2 is a transmemb rane spanning receptor-like protein, which is struc-
turally related to the epidermal growth factor receptor, and blockade of HER2 signaling by
trastuzumab has been shown to inhibit the proliferation of human tumor cells that overex-
press HER2 both in vitro and in vivo (Lee, 1995; Carter et al., 1992). Herceptin is indicated
for the treatment of patients with metastatic breast and gastric cancer whose tumors overex-
press HER2. The recom mended dosage scheme consists of a trastuzumab loading dose and
subsequent maintenance doses administered until progression of disease (EMEA scientific
discussion) (EMA, 2011). In cross-reactivity studies with frozen human or cynomolgus mon-
key tissues, trastuzumab and its parent murine antibody, muMAb4D5, showed similar pat-
terns of immunoreactivity. They were both reactive in no rmal tissues with membrane
staining in a subset of epithelial cells including squamous ep ithelium of exocervix, skin,
esophagus, urothelium of the bladder, and tonsil. Epithelial cells of different organs showed
positive membrane staining in breast acinar and ductal cells, endocervical glands, esophageal
glands, epithelial cells of the renal tubules, and epithelial cells lining the gastrointestinal tract,
including pancreas and salivary glands. High sequence homology of human ErbB2 (encoded
by the same gene as HER2) with cynomolgus monkey ErbB2, and compatible/parallel bind-
ing patterns in human and rhesus monkey tissue screens indicated that monkey was an
acceptable toxicology species. The tissue cross-reactivity and nonclinical PK studies and
the demonstrated specificity of muMAb4D5 for HE R2, support the conclusion that trastuzu-
mab recognized monkey HER2. Rhesus monkeys were used for single-dose and short-term
repeat-dose studies (treatment periods 6 months), whereas cynomolgus monkeys were
used for longer term (3 months) repeat-dose studies. Trastuzumab did not cross-react
with the corresponding rodent receptor (p185neu) (Hu et al., 2002).
Trastuzumab-induced complement-dependent cytotoxicity (CDC) was not observed,
which is likely due to the presence of membrane-associated complement regulatory proteins
such as CD35 (complement receptor 1, CR1), CD55 (decay accelerating factor, DAF), or CD46
(membrane cofactor protein, MCP). However, trastuzumab did support robust ADCC
against HER2-overexpressing cells, a desired effect of the molecule. Trastuzumab-
dependent ADCC was mediated by FcgRIII on natural killer cells and monocytes. Interaction
11. Biological therapies for cancer374
III. Specialty biologics and indications
with this low-affinity Fcg receptor is avidity driven; opsonization of tumor cell targets with
trastuzumab was required for activity. The avidity component of trastuzumab-dependent
ADCC contributes to the safety profile of the antibody in patients; HER2-overexpressing tu-
mor cells would likely be preferentially targeted for ADCC rather than tissues with normal
levels of HER2 (Yuan et al., 1996; Hu et al., 2002).
The pharmacokinetics observed at lower doses of trastuzumab in monkey is consistent
with specific, saturable binding. The induction of antibodies against trastuzumab was a
rare event in the monkeys. Neutralizing antibodies were detected from weeks 5 to 26 in
one low-dose female cynomolgus monkey out of a total of 84 animals (Yuan et al., 1996;
Hu et al., 2002). However, it is recognized that the sensitivity of the detection system of anti-
trastuzumab antibodies could be compromised by the presence of trastuzumab in the serum
samples of the monkeys.
Single-dose acute toxicology studies were undertaken using IV bolus administration in
mice at 0, 9.4, 47, and 94 mg/kg and in rhesus monkeys at 0, 4.7, 23.5, and 47 mg/kg. It is
unclear why a mouse study was performed, but the timing of the study was during the in-
fancy of biologics development and could have been performed for formulation testing as
multiple formulations were used. The absence of toxicity of several different preparations
and formulations of trastuzumab was demonstrated, as measured by standard in-life param-
eters including clinical chemistry and also by macro- and microscopic examination of stan-
dard organs/tissues. The NOELs were determined as the highest doses tested , 94 and
47 mg/kg in mice and monkeys, respectively (Yuan et al., 1996; Hu et al., 2002).
The repeat-dose toxicity evaluation of trastuzumab included a 4-week study in rhesus
monkeys and 12- (twice weekly) and 26-week (weekly) studies in cynomolgus monkeys. In
all three studies, trastuzumab was administered intravenously and was well tolerated up
to the highest dose that could be delivered (25 mg/kg), with the only noteworthy observa-
tions concerning injection site reactions in the rhesus monkey. There were no other toxicolog-
ically significant findings (Yuan et al., 1996; Hu et al., 2002).
Because of the lack of relevance of conventional species (rat, rabbit) used for reproductive
toxicity studies, reproductive studies were conducted in the cynomolgus monkey. In a study
of female cynomolgus monkeys given trastuzumab at doses up to 25 times the weekly human
maintenance dose of 2 mg/kg as daily intravenous injections for 4 days followed by twice-
weekly admin istration for the duration of the dosing period, no alterations in menstrual
cyclicity or sex hormone profiles, and no trastuzumab-related embryotoxicity or effects on
fetal development were observed. Pregnancy did not appear to affect maternal exposure to
trastuzumab. The labeling of trastuzumab included the reproductive toxicity study results
but also contained a statement indicating “the significance of the rodent form of the HER2
receptor in normal embryonic development and the embryonic death in mutant mice lacking
this receptor” was relevant to human risk assessment (Yuan et al., 1996; Hu et al., 2002;
Fanale et al., 2005). When trastuzumab was administered during the period of organogenesis,
fetal serum trastuzumab concentrations ranged from 10% to 19% of maternal values (Hu
et al., 2002). Administration during the last trimester was associated with trastuzumab fetal
serum concentrations of approximately 33% of maternal concentrations. The difference in
fetal serum trastuzumab concentrations obtained in the early and late gestational periods
may be attributable to the time between trastuzumab administration and maternal/fetal
blood sampling (e.g., samples were obtained 50 days or 2 days after the final trastuzumab
Bevacuzimab 375
III. Specialty biologics and indications
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