Download Inertly labeled monoclonal antibodies for PET and optical imaging

Chapter 1
General introduction
General introduction
Antibody-based therapy has become one of the most important and successful
therapeutic modalities to treat cancer. The first application was just 20 years ago,
but the concepts are much older. Antibody-based therapy is actually one of the oldest treatments for cancer patients. Back in 1895, two French researchers described
a serotherapy in which they treated patients with antisera derived from animals that
were injected with cancer cells of the patient themselves1. In some of the patients
improvements were observed, however, severe immunological reactions occurred
in most cases. The antisera they used consisted of a large variety of antibodies
directed towards different antigens. Some of these antigens are expressed not only
on malignant cells, but also on normal cells, which caused the immunological
reactions. If a method could be developed to produce antibodies directed against
one specific antigen, a possible treatment against cancer and many other diseases
could be envisioned. This was the foundation of the ‘magic bullet’ hypothesis,
proposed a decade later by Paul Ehrlich2. His theory implied that if a compound
could be made that selectively targets a disease, then a toxin for that organism
could be delivered along with this selective agent.
Antibody structure and function
Natural human antibodies are large protein molecules produced by plasma cells,
a specific type of white blood cells, and are used to identify and neutralize foreign
organisms or molecular structures, called antigens. The structure of antibodies was
discovered in the late 1960s3,4. Antibodies consist of two identical heavy chains
that are interconnected forming a Y-shape and two light chains that form extra legs
to the top of the Y (Figure 1). Heavy and light chains both have a constant and a
variable region. Heavy chains determine the class of the antibody, of which there
are five types in mammals. The tail of the Y is indicated as the Fc part and the
variable regions are the upper parts of the heavy and light chains that form the two
upper legs (Fab-part) of the Y. The two light chains are identical and also consist
Figure 1 - Schematic representation of a mAb.
9
1
10
Chapter 1
of a bottom constant region and a top variable region. Antigen binding occurs at
the variable part. When the antibody is bound to the antigen by the Fab (Fragment,
antigen binding) region on the top, the tail, called Fc (Fragment, crystallization)
region makes it possible to initiate the further immune response that will remove
the antigen from the system.
Development of monoclonal antibodies
From the early 1900s on, many researchers tried to find a method to develop antibodies directed against one specific antigen, i.e. monoclonal antibodies (mAbs). In
1951, Henri Kunkel investigated cells from multiple myeloma patients and discovered that the malignant plasma cells produce antibody molecules with one single
specificity instead of several5. However, these cells produce antibodies at random,
and researchers had no method to find out against which antigen the antibody
was directed. In the late 1960s, researchers began to develop methods to isolate
and propagate B-cells that only produce one specific antibody directed against
one specific antigen6. The limitation of these methods was the restricted lifespan
of the B-cells and the small amount of antibodies produced. In 1975, Köhler and
Milstein succeeded in fusing a mouse B-cell, producing a specific and well-known
antibody, with a mouse myeloma cell7. This method was called the ‘hybridoma
technique’. By using this hybridoma technology they obtained an immortal cell
line, which was capable of very high and pure production of one specific antibody.
This method to develop specific antibodies did not immediately lead to convincing clinical successes, since the antibodies were derived from mice. Application
of murine mAbs met a few limitations: they are quickly removed from the blood,
inflammatory reactions can occur, and human anti-mouse antibodies (HAMAs) can
develop. When HAMAs are present, patients cannot receive multiple doses of the
mAb, because of anaphylactic reactions that might occur8. By genetic engineering,
chimeric antibodies were developed that are partially mouse and partially human.
The DNA encoding the variable parts of the murine heavy and light chains are
fused to the DNA encoding the constant regions of the human heavy and light
chains, and these fusion constructs are brought into cultured cells for chimeric
antibody expression. This engineering also allows the design of antibodies of a
particular isotype such as IgG1 or IgG4. With this approach immune reactions
directed against the Fc part of the mAb can be avoided, while the antigen binding
part is not altered. As a next step it became possible to replace most of the murine
heavy and light chains by the human heavy and light chains, with just the antigen
binding parts of murine origin remaining. These mAbs are called “humanized”.
Nowadays techniques are available to develop also fully human mAbs, and in some
approaches the use of mice or other animals is no longer needed. These methods
General introduction
also enable the engineering of a variety of antibody fragments, such as: diabodies,
minibodies, affibodies and nanobodies9. With the increase in mAb-development,
rules for the nomenclature also have been set. All mAbs should have -mab as a
suffix, and are preceded by the origin-determination: -o for mouse, -u for human,
-xi for chimeric, -xizu for chimeric/humanized, and -zu for humanized.
First mAbs and their mechanisms of action
All of the current FDA-approved anti-cancer mAbs are immunoglobulins G (IgGs),
with a variety of mechanisms of action, not always predictable and not always
known for a new mAb. Often these mechanisms work in parallel, depending on
the targeted molecule and the isotype of the mAb. Mechanisms of action include:
(i) inhibition of cell signaling, (ii) induction of apoptosis, (iii) antibody-dependent
cellular cytotoxicity (ADCC), and (iv) complement-dependent cytotoxicity (CDC).
While the first two mechanisms are direct effects of the antibody, the next two
are immune-mediated responses. In addition a mAb can serve as a carrier for a
toxic agent. The delivery of a toxic agent is central part of this thesis and will be
discussed later.
The different mechanisms of action are explained hereafter in more detail: (i) in
the inhibition of cell signaling the antibody has an antagonistic effect by binding a
membrane-bound receptor, often a growth factor receptor that shows and increased
level of expression in tumor cells. This either prevents the growth factor from binding to its receptor or interferes with dimerization of the receptor. The result is a
decreased signaling in the downstream pathway, leading to inhibition of unwanted
cell proliferation and, ideally, cell death. The antibody could also target the growth
factor itself, and as a result binding to the receptor cannot occur and cell growth is
halted; (ii) antibodies that induce apoptosis have an agonistic effect, and binding
to a receptor directly triggers apoptotic signaling in the tumor cells. The Fc-region
of IgGs mediates immune-mediated responses: after binding to a target molecule,
the Fc-regions engage effector cells of the immune system that subsequently kill
the tumor cells; (iii) ADCC is induced by binding of the Fc-region of the mAb to
the FcγRIIIa (CD16) receptors on e.g. natural killer (NK) cells, after which NK cells
are activated and release cytolytic granules that cause cell death; and (iv) CDC is
induced when the Fc-region of the mAb binds to soluble complement factor C1q.
This triggers the complement cascade that ultimately leads to cell death.
Clinically approved mAbs
The developments of the last years have led to 17 currently FDA- and European
Medicines Agency (EMA)-approved mAbs (Table 1). Some of these are exploited as
naked mAbs and some as antibody-conjugates.
11
1
12
Chapter 1
Table 1 - FDA- and EMA-approved mAbs for use in oncology, adapted from Glassman & Balthasar18
Name
Marketed by
Class
Target
First approved
indication
Reported
mechanisms of
action
Approval
year
Rituximab
(Rituxan)
Biogen Idec./
Genentech
Chimeric
IgG1
CD20
Non-Hodgkin’s
Lymphoma
ADCC, CDC,
Induction of
Apoptosis
1997
Trastuzumab
(Herceptin)
Genentech
Humanized
IgG1
HER2
Breast Cancer
Signal Inhibition, 1998
ADCC
Alemtuzumab
(Campath)
Sanofi-Aventis
Humanized
IgG1
CD52
B cell Chronic
Lymphocytic
Leukemia
CDC, Induction
of Apoptosis
2001
Ibritumomab
tiuxetan
(Zevalin)
Biogen Idec
Murine IgG1 CD20
Non-Hodgkin’s
Lymphoma
Radioisotope
Delivery (90Y)
2002
Tositumomab
(Bexxar)
GlaxoSmithKline
Murine
IgG2a
CD20
Non-Hodgkin’s
Lymphoma
Radioisotope
Delivery (131I),
ADCC, CDC,
Induction of
Apoptosis
2003
Cetuximab
(Erbitux)
Bristol-Myers
Chimeric
Squibb/Eli Lilly IgG1
EGFR
Signal Inhibition, 2004
Squamous Cell
Carcinoma of the ADCC, CDC
Head and Neck
Bevacizumab
(Avastin)
Genentech
Humanized
IgG1
VEGF
Colorectal
Cancer
Signal Inhibition
Panitumumab
(Vectibix)
Amgen
Human IgG2 EGFR
Colorectal
Cancer
Signal Inhibition, 2006
ADCC
Ofatumumab
(Arzerra)
Genmab/GSK
Human IgG1 CD20
Chronic
Lymphocytic
Leukemia
ADCC, CDC
2009
Denosumab
(Xgeva)
Amgen
Human IgG2 RANKL Bone Metastases
Signal Inhibition
2010
Ipilumumab
(Yervoy)
Bristol-Myers
Squibb
Human IgG1 CTLA-4 Metastatic
Melanoma
Signal Inhibition
2011
Brentuximab
vedotin
(Adcetris)
Seattle
Genetics
Chimeric
IgG1
CD30
Hodgkin
Lymphoma
ADC
2011
Pertuzumab
(Perjeta)
Genentech
Humanized
IgG1
HER2
Breast Cancer
Signal Inhibition, 2012
ADCC
Trastuzumab
emtansine
(Kadcyla)
Genentech
Humanized
IgG1
HER2
Breast Cancer
ADC, Signal
Inhibition,
ADCC
2013
Obinituzumab
(Gazyva)
Genentech
Humanized CD20
IgG1; Glycoengineered
Chronic
Lymphocytic
Leukemia
ADCC, CDC,
Induction of
Apoptosis
2013
Pembrolizumab
(Keytruda)
Merck Sharp & Humanized
Dohme Corp
IgG4
PD-1R
Metastatic
Melanoma
Signal Inhibition
2014
Nivolumab
(Opdivo)
Bristol-Myers
Squibb
Human IgG4 PD-1R
Metastatic
Melanoma
Signal Inhibition
2014
2004
General introduction
Naked mAbs
In 1997, rituximab was the first mAb in oncology that was approved by the US
Food and Drug Administration (FDA). It is directed against the CD20 antigen and
used for patients with non-Hodgkin’s lymphoma (NHL). Hematologic malignancies are relatively easy to target, because the cells are in close connection with the
blood stream and therefore directly accessible for mAb-therapy. CD20 is highly
expressed on normal B cells and B-cell lymphoma cells and is not shed into the
plasma. In vitro, no internalization after binding of rituximab is observed, but in
vivo internalization cannot be excluded. Although also normal B cells are cleared
by rituximab treatment, this seems to have no effect on the immune system, since
no increase in the incidence of infections is observed in these patients10. Upon
antibody binding, B-cell proliferation is directly inhibited, nuclear DNA fragmentation is induced and this leads to cell death by apoptosis11. After being a decade on
the market, rituximab still makes 5 billion dollar sales per year12.
One year after the approval of rituximab, a mAb targeting solid tumors was approved: trastuzumab. This humanized mAb is directed against human epidermal
growth factor receptor 2 (HER2), which is expressed by many epithelial tissues and
over-expressed in 20-30% of breast cancers. Trastuzumab binds to the extracellular
domain IV of the HER2 receptor and blocks activation of the downstream signaling
cascade. Other indicated effects are prevention of HER2-receptor dimerization,
increased endocytotic destruction of the receptor, inhibited shedding of the extracellular domain, and activation of the immune system by ADCC13. Trastuzumab
is used for treatment of metastatic HER2-positive breast cancer and as adjuvant
therapy for other HER2-positive breast tumors.
Alemtuzumab became approved in 2001, and is an anti-CD52 mAb used for
patients with B-cell chronic lymphocytic leukemia. It exerts anti-tumor activity
by ADCC. In 2012, alemtuzumab was withdrawn from the market and was relaunched in 2014 as a treatment for multiple sclerosis at a much higher price.
Cetuximab, approved in 2004, is directed against the epidermal growth factor
receptor (EGFR = HER1) and was first used in advanced colorectal cancer. It is only
effective for tumors that express EGFR and have a wild type KRAS gene. Like trastuzumab, cetuximab blocks the downstream signaling cascade after binding with
high affinity to the receptor. KRAS is downstream in the signaling cascade, when
mutated it causes uncontrolled cellular proliferation and as it acts downstream of
EGFR it makes the tumor cell insensitive for cetuximab treatment. It also triggers
ADCC and CDC. Currently, cetuximab is registered for the treatment of metastatic
colorectal cancer (mCRC), metastatic non-small cell lung cancer (mNSCLC) and
head-and-neck squamous cell carcinoma (HNSCC).
13
1
14
Chapter 1
Bevacizumab, also approved in 2004, is a mAb directed against vascular endothelial growth factor (VEGF), a growth factor associated with angiogenesis and
overexpressed in many solid tumors. Bevacizumab binds to VEGF, thereby causing
inhibition of angiogenesis. When tumors grow larger, the cells become hypoxic
and start releasing VEGF and other angiogenic factors. This makes bevacizumab
suitable for use in many different solid tumor types. Currently it is approved for
treatment of patients with mCRC, NSCLC, metastatic breast cancer, metastatic
renal cell cancer, prostate cancer, and glioblastoma14.
Panitumumab, approved in 2006, is an anti-EGFR mAb, like cetuximab, used in
the treatment of mCRC and also requires wild type KRAS to be effective. In contrast
to cetuximab, which is a chimeric IgG1 and therefore ideally suited for ADCC
activity, panitumumab is a human IgG2 mAb, less active in ADCC. However, clinical data do not show differences in the efficacy of these two anti-EGFR mAbs in
colorectal cancer 15, while there is a difference in HNSCC.
Ofatumumab, approved in 2009, targets CD20 like rituximab and is approved
for use in treatment of chronic lymphocytic leukemia (CLL). The binding and immunological properties of ofatumumab differ from those of rituximab16, therefore
it might be a suitable mAb to treat rituximab-refractory patients. In 2012, a clinical
study showed a modest efficacy of ofatumumab treatment in rituximab-refractory
patients17.
Denosumab, approved in 2010, targets less frequent occurring tumors. It is
directed against the receptor activator of nuclear factor kappa-B ligand (RANKL), a
surface molecule on osteoclasts, and is used for treatment of bone metastases from
solid tumors and for unresectable giant cell bone tumors18.
Ipilimumab, the first immune checkpoint mAb (elaborated in the part ‘Other next
generation mAbs’) approved in 2011, is used for treatment of metastatic melanoma
and is directed against cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), a
protein receptor that down-regulates the immune system19.
Pertuzumab, approved in 2012, is also directed against HER2 and used to treat
patients with metastatic breast cancer. Pertuzumab targets a different motif in HER2
than trastuzumab: it binds domain II of the extracellular domain of HER2, and is
more effective in preventing receptor heterodimerization with EGFR, HER3, and
HER4, and subsequent cell signaling. Preclinical studies showed synergy between
trastuzumab and pertuzumab, providing possibilities for combination therapy20.
Therefore, a phase II clinical study was performed, which showed that the addition
of pertuzumab to the standard trastuzumab and docetaxel combination treatment
resulted in a significant clinical benefit21.
General introduction
Conjugated mAbs
Using mAbs as delivery vehicles of toxic agents is one of the most promising applications in antibody therapy. During the earliest mAb research this approach was
tested because of the initial lack of successes with naked therapeutic murine mAbs.
Delivery of toxic payloads can be divided in three categories: radioimmunoconjugates, immunotoxins and antibody-drug conjugates (ADCs). The development
of mAbs coupled to radioactive or toxic compounds makes the chemistry more
complicated and demands a higher standard of safety measurements: (i) conjugates preferably have exactly the same pharmacokinetics as their non-conjugated
counterparts; (ii) biological half-life and binding properties to the target should not
be altered after conjugating an additional group to the mAb; (iii) in vivo stability of
the chemical bond between the payload and the mAb is also of major importance,
since it is undesirable that some of the toxic molecules are released from the conjugate before they reach the tumor site.
Radioimmunoconjugates are mAbs with a radionuclide or radionuclide-containing compound attached to it. When the mAb binds to the tumor cell, the radiation
damage causes cell death of the targeted cell and possibly the surrounding cells,
depending on the radiation energy of the radionuclide. For use in this therapeutic
setting, the radionuclide needs to have a physical half-life long enough to allow
the mAb to reach the tumor and have an α- or β– -decay. The two radionuclides
used in FDA-approved radioimmunoconjugates are 131I and 90Y, with half-lifes of
8.02 days and 2.66 days, respectively. 90Y emits high-energy β’s that have a mean
range in tissue of 2.7 mm, and is therefore most suitable for large tumors; 131I emits
β’s with lower energy, the mean tissue range is 0.8 mm, and is better suited for
treatment of smaller tumors22. The radionuclide is coupled directly to the tyrosines
of the mAb (131I) or via a chelate (90Y). The two radioimmunoconjugates that made
it to approval are ibritumomab tiuxetan (90Y) in 2002 and tositumomab (131I) in
2003 (see Table 1). Both antibodies are of murine origin with CD20 as the target.
They are used for treatment of patients with NHL, which is attractive since these
cells are sensitive for radiation. Nevertheless, unlabeled mAbs like rituximab and
its successors are still favored in clinical use, since working with radiopharmaceuticals always requires extra precautions for the patient and physician. In recent
years, new radioimmunoconjugates have not been approved for clinical use. Main
reasons are the inadequate targeting of many tumor types for radioimmunotherapy
and the previously mentioned extra precautions that need to be implemented for
transportation and clinical use when working with radiopharmaceuticals.
Immunotoxins have a catalytic toxin as therapeutic entity. These toxins are
enzymes of bacteriological origin or other types of pathogens. After endocytosis,
the cleaved toxins cause termination of protein synthesis, which results in cell
15
1
16
Chapter 1
death by apoptosis. A complication in the development of immunotoxins is that
the toxins are of non-mammalian origin, which causes a reaction of the human
immune system in patients. Neutralizing antibodies are produced against the toxin,
limiting the number of treatments23. Also, most toxins have binding sites for surface
molecules expressed by normal tissues, and these binding sites have to be modified
before the toxins can be used therapeutically. In addition, therapy studies revealed
that efficacy could be impeded by cellular resistance mechanisms24. While immunotoxins have a lot of potential, a large effort in optimization is required before
they can be successfully brought to the clinic.
ADCs are currently the most thoroughly explored immunoconjugates. For the last
20 years researchers have tried to develop the perfect combination of a highly toxic
payload, linked through a stable linker to a specifically and selectively targeting
mAb. Early studies tested classical chemotherapeutic compounds, such as doxorubicin, coupled to a mAb, but these conjugates failed to show therapeutic efficacy25.
These failures can be explained by the small amount and relatively low potency
of the toxic compound that is released in the tumor. Compared to a single injection of the free toxic compound, the amount of toxic compound that is delivered
to the tumor is much lower when coupled to a mAb26. Therefore, the ideal ADC
allows the application of highly toxic compounds, much more toxic than current
chemotherapeutics, that otherwise have a therapeutic window which is too narrow
to allow their use as free drug27. This also requires stable conjugation to avoid
systemic toxicity by release of the toxic compound in the circulation, but once
internalized the drug should be released to be able to kill the tumor cell. Only
then, the desired therapeutic effect will be observed.
The toxic compounds used for ADCs can be divided in two main categories:
DNA-damaging agents and microtubule inhibitors. The most studied toxins are
tubulin inhibitors derived from maytansine, maytansoids, and the auristatins,
synthetic analogues derived from the natural occurring dolastatin 10, and the
DNA-damaging agent calicheamicin28. The development of synthetic versions of
these natural occurring toxins provided the possibility of adding specific chemical groups, which enables easy coupling to linkers while keeping toxicity in the
nanomolar range.
Gemtuzumab-ozogamicin was in 2000 the first FDA approved ADC and used
for the treatment of acute myeloid leukemia (AML) (Table 1). This IgG4 mAb is
directed against CD33, a surface glycoprotein, and the cytotoxic antibiotic calicheamicin is linked to this mAb by a hydrazone (pH labile) linker. The IgG4-isotype
was used because it does not induce ADCC activity and related cytokine release.
Gemtuzumab-ozogamicin was used as single agent for patients over 60 with
General introduction
CD33-positive AML in first relapse or those that are not considered candidates
for cytotoxic chemotherapy29. However, 10 years after its approval, gemtuzumabozogamicin was voluntarily withdrawn from the market due to the lack of clinical
benefit and unacceptable systemic cytotoxicity30. The poor results with this first
approved ADC demonstrate the importance of getting more knowledge on the in
vivo behavior of ADCs, e.g. in animal models.
A decade after the approval of gemtuzumab-ozogamicin, the second ADC was
approved for clinical use: brentuximab vedotin (Table 1), directed against CD30
and used to treat patients with Hodgkin’s lymphoma. The drug used in this ADC is
the auristatin derivative monomethyl auristatin E (MMAE), coupled to the mAb via
a cleavable peptide (valine-citrulline) linker. Brentuximab vedotin was developed
after early-phase patient data revealed only modest clinical activity of the naked
mAb, originally named cAC10. In 2003, the ADC was developed under the name
cAC10-vcMMAE/SGN-35, but the ADC was renamed brentuximab vedotin in
2008 when clinical studies were started.
The next approved ADC was ado-trastuzumab-emtansine in 2013 (Table 1).
The ADC consists of trastuzumab and the maytansine derivate DM1, coupled via
a thioether linker proclaimed to cause the release of the drug upon proteolytic
degradation only. The thioether linked ADC gave the better therapeutic effect, since
the release of the toxic compound in the circulation was minimized compared
to other, less stable, hydrazone linkers31. Ado-trastuzumab emtansine is approved
for treatment of patients with metastatic breast cancer who previously received
trastuzumab and a taxane, separately or in combination32.
At the moment more than 30 ADC’s are in early- or late-stage clinical development28. The ADCs in late stage clinical development all contain the previously
mentioned toxic compounds (Table 2).
Researchers tried to learn from the withdrawal of gemtuzumab ozogamicin and
developed a closely related ADC: inotuzumab ozogamicin, the same drug and
linker, coupled to CD22, which is another surface glycoprotein that can be used
to treat patients with AML/NHL. The naked CD22 antibody showed no clinical
efficacy, but is readily internalized, which makes it ideal for use in an ADC, as was
confirmed by preclinical and early clinical studies33. Clinical phase III studies with
izotuzumab ozogamicin in combination with rituximab are currently ongoing.
Pinatuzumab vedotin is also directed against CD22 and contains an auristatin
toxic compound, like in the approved brentuximab vedotin. It is also a potential
competitor of rituximab, the current standard of care for treatment of NHL in combination with chemotherapy, as was shown in preclinical in vivo efficacy studies34.
17
1
18
Chapter 1
Therefore, a phase II study was started that combines pinatuzumab vedotin with
rituximab35, to find a possible treatment for rituximab-refractory patients.
For treatment of solid tumors, glembatumumab vedotin and lorvotuzumab
mertansine are currently in phase II studies. Glembatumumab vedotin contains a
mAb directed against glycoprotein non-metastatic melanoma protein B (GPNMB),
which is mainly expressed in breast cancers and melanomas. The toxic MMAE is
conjugated to the mAb via a cleavable peptide linker, like in brentuximab vedotin.
Clinical studies with glembatumumab vedotin for treatment of both advanced
melanoma36 and breast cancer37 show promising results. Lorvotuzumab mertansine
is composed of an anti-CD56 antibody, linked via a cleavable disulfide linker to the
maytansinoid DM1. CD56 is the neuronal cell adhesion molecule and is expressed
by various tumor types. Lorvotuzumab mertansine has shown efficacy in combination therapy in multiple myeloma patients38 and shows potential in small-cell lung
cancer (SCLC) xenograft models39. These combined results were promising enough
to immediately start with the phase II clinical trial for treatment of SCLC35.
Despite increasing popularity of ADCs, it is fair to state that the development of
ADCs is still at its infancy. At this moment, most ADCs under clinical development
contain maytansines or auristatins as the toxic payload (Table 2). It is not totally
clear whether releasable, cleavable or non-cleavable linkers are best suited for
appropriate drug delivery to the tumor. To get better understanding of optimal ADC
design, in vivo tracking of ADCs would be very informative.
Table 2 - Clinical-stage ADC pipeline, adapted from Mullard35
ADC
Lead
Lead indications
Target
Payload
Phase
Inotuzumab
ozogamicin (CMC544)
Pfizer
Aggresive nonHodgkin’s lymphoma;
ALL
CD22
Calicheamicin
III
RG-7596
Genentech
DLBCL and follicular
non-Hodgkin’s
lymphoma
CD79b
MMAE
II
Pinatuzumab
vedotin (RG-7593)
Genentech
DLBCL and follicular
non-Hodgkin’s
lymphoma
CD22
MMAE
II
Glembatumumab
vedotin
Celldex
Breast cancer
GPNMB
MMAE
II
SAR-3419
Sanofi
DLBCL; ALL
CD19
DM4
II
ImmunoGen
Lorvotuzumab
mertansine (IMGN901)
Small-cell lung cancer CD56
DM1
II
BT-062
BioTest
Multiple myeloma
CD138
DM4
II
PSMA-ADC
Progenics
Prostate cancer
PSMA
MMAE
II
General introduction
Table 2 (continued)
ADC
Lead
Lead indications
Target
Payload
Phase
ABT-414
AbbVie
Glioblastoma; NSCLC; EGFR
solid tumor
Not disclosed
I/II
Melatuzumab
doxorubicin
Immunomedics
CLL; multiple
myeloma; nonHodgkin’s lymphoma
CD74
Doxorubicin
I/II
IMMU-132
Immunomedics
Solid tumor
Irinotecan
TACSTD2
metabolite
(also known as
TROP2 or EGP1)
I
LabetuzumabSN-38
Immunomedics
Cancer; colorectal
cancer
CEA (also known Irinotecan
as CD66e)
metabolite
I
IMGN-853
ImmunoGen
Ovarian tumor; solid
tumor
Folate receptor 1 DM4
I
IMGN-529
ImmunoGen
B cell lymphoma;
CLL; non-Hodgkin’s
lymphoma
CD37
DM1
I
RG-7458
Genentech
Ovarian tumor
Mucin 16
MMAE
I
RG-7636
Genentech
Melanoma
Endothelin
receptor ETB
MMAE
I
RG-7450
Genentech
Prostate cancer
STEAP1
MMAE
I
RG-7600
Genentech
Ovarian tumor;
pancreatic tumor
Not disclosed
Not disclosed
I
RG-7598
Genentech
Multiple myeloma
Not disclosed
Not disclosed
I
RG-7599
Genentech
NSCLC; ovarian tumor Not disclosed
Not disclosed
I
SGN-CD19A
Seattle Genetics ALL; aggressive nonHodgkin’s lymphoma
CD19
MMAE
I
Vorsetuzumab
mafodotin
Seattle Genetics Non-Hodgkin’s
lymphoma; renal cell
carcinoma
CD70
MMAF
I
ASG-5ME
Agensys
Cancer; pancreatic
tumor; stomach tumor
SLC44A4
(AGS-5)
MMAE
I
ASG-22ME
Agensys
Solid tumor
Nectin 4
MMAE
I
AGS-16M8F
Agensys
Cancer; renal cell
carcinoma
AGS-16
MMAF
I
MLN-0264
Millennium
Gastrointestinal tumor; Guanylyl
solid tumor
cyclase C
MMAE
I
SAR-566658
Sanofi
Solid tumor
Mucin 1
DM4
I
AMG-172
Amgen
Cancer; renal cell
carcinoma
CD70
Not disclosed
I
AMG-595
Amgen
Glioma
EGFRvIII
DM1
I
BAY-94-9343
Bayer
Cancer; mesothelioma Mesothelin
DM4
I
ALL, acute lymphoblastic leukemia; CEA, carcinoembryonic antigen; CLL, chonic lymphoblastic leukemia; DLBCL, diffuse large B cell lymphoma; GPNMB, glycoprotein NMB; MMAF, monomethyl auristatin
F; NSCLC, non-small-cell lung cancer; PSMA, prostate-specific membrane antigen; STEAP1, six-transmembrane epithelial antigen of prostate 1; TACSTD2, tumour-associated calcium signal transducer 2.
19
1
20
Chapter 1
Other next generation mAbs
While in the ADC-development the focus is on improving the linker/toxic compound chemistry, the mAb itself is also undergoing development, contributing to
next generation mAbs. Glycoengineered mAbs are modified in their glycosylation
pattern in the Fc-region to enhance ADCC and CDC, via improved binding to
Fcγ receptors on immune effector cells. This creates a more potent naked mAb,
compared to a normal IgG1, with ofatumumab40 as the first successful example of
this strategy and obinituzumab the second that was approved in 2013 (Table 1).
A new and exciting development is the clinical application of mAbs that inhibit immune-checkpoints. When an immune response is elicited, also a negative
feedback mechanism is activated to dampen the response, and these mechanisms
are indicated as immune-checkpoints. These mechanisms are protective, but
hamper effective anti-tumor immune responses. In fact tumor cells can exploit
these mechanisms to counteract immune responses41. Cytotoxic T-lymphocyteassociated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have
proven to be important immune-checkpoint proteins and promising therapeutic
targets for mAbs. The previously mentioned ipilimumab is directed against CTLA-4
and nivolumab and pembrolizumab are directed against PD-1. Nivolumab and
pembrolizumab, approved in 2014 (Table 1), reactivate T cells by blocking APC
(antigen presenting cell) inhibition of T cells or by blocking tumor cells, which often inactivate T cells in the microenvironment via their overexpressed PD-1 ligands
(e.g. PD-L1)42. An advantage of these immune-checkpoint inhibiting mAbs is that
they can be beneficial in many types of tumors, hematological and solid.
Multi-specific antibodies are able to recognize more than one target molecule, by
exploiting single heavy chain-light chain pairs from antibodies with distinct specificities. The different pairs can be linked to form two arms with different specificity
within a single IgG molecule. MEHD7945A, currently in phase II clinical trials, is
directed against EGFR as well as HER3, which are closely related receptors, and
shows promising results in the treatment of multidrug resistant tumors43.
Besides intact mAbs and antibody fragments, nanobodies included, advances in
genetic engineering have led to the development of several non-immunoglobinlike binding molecules. They have antibody-like binding properties, but completely
lack the antibody scaffold; and the novel structures are designed as derivatives
of naturally occurring substrates or proteins. Anticalins, affibodies, adnectins,
designed ankyrin repeat proteins (DARPins) and avimers are among the most
developed non-Ig-based protein scaffolds44. Claimed advantages of these ligands
are their small size, max. 200 amino acids, and the lack of immunogenicity. Adnectin CT-322 and DARPin AGN150998/MP0112 are currently in clinical phase II
development.
General introduction
Nuclear imaging in mAb development and applications
The continuous progress in the development of mAbs and mAb conjugates demands
for accurate, preferably quantitative, methods to monitor the in vivo behavior of
these potentially toxic molecules. Therefore, not only mAbs have been in development for therapeutic exploitation the last decades, but many researchers have put
as much effort in the development of diagnostic mAbs as well.
Most commonly used diagnostic mAbs for imaging purposes consist of a mAb
carrying a radioactive isotope for use in single photon emission computed tomography (SPECT) or positron emission tomography (PET)45. The quality of gamma
cameras as well as PET-scanners in terms of sensitivity and resolution has improved
over the last years, causing an increase in mAb-imaging. However, PET imaging
has a higher sensitivity and more accurate quantification than SPECT.
Immuno-PET, imaging of mAbs labeled with positron emitters, can provide
whole body images, thereby revealing important information both about the tumor
and possible metastases. Also the level of antigen expression in tumor and other
tissues can be determined, indicating the tumor-specificity of the mAb: crucial
knowledge for therapeutic potential of the mAb. Immuno-PET can also be used to
optimize mAb dose scheduling and to track changes in tumor biology over time,
including assessments of the antigen expression before and after therapy. Furthermore, immuno-PET provides the possibility to select patients for therapy, thereby
increasing therapeutic efficacy and decreasing adverse events.
The radionuclides that can be used for immuno-PET, need to have a physical halflife that matches the biological half-life of the mAb. Since almost all approved mAbs
and mAbs in clinical development are intact mAbs with a half-life of several days, 124I
(t1/2=100.3 hours) and 89Zr (t1/2=78.4 hours) are attractive candidate radionuclides. For
both isotopes labeling procedures have been developed that ensure safety-controlled
production for human use all over the world9. Conjugation with 124I is relatively easy,
only an oxidizing agent is necessary to enable the direct labeling reaction of 124I to
the tyrosine moiety of the mAb. Labeling mAbs with 89Zr requires premodification of
the mAb, which implies the introduction of an extra group - a chelator - to be able
to attach the metal radionuclide in a stable way. The first method used for clinical
immuno-PET was published in 200346 and required a two steps conjugation. The
same group published an improved version in 2010 with a single step conjugation47.
An important reason to choose between 124I or 89Zr is the nature of the mAb, whether
it is internalizing or not. Iodine is released from the cells after internalization, while
the zirconium is residualizing and remains in the cell after internalization. The latter
makes 89Zr the radionuclide of choice for many immuno-PET applications.
Many successful preclinical and clinical studies with 124I- and 89Zr labeled mAbs
have been performed and are described in detail in comprehensive reviews9, 48-51.
21
1
22
Chapter 1
Near-infrared imaging in mAb applications
Although immuno-PET has many advantages for whole body imaging, resolution
is limited and real-time imaging is impossible. Photoimmunodetection, in which
mAbs are labeled with fluorescent dyes, might have a complementary clinical
potential to immuno-PET. It allows high-resolution, real-time, dynamic imaging
of superficial tissue layers at the cellular level, without radiation burden to the
patient. Therefore, it might be ideal for confirmation of mAb binding to subsets
of cells. In addition, it might be a powerful clinical tool for the delineation and
characterization of early-stage or residual disease, for example of cancer during
surgery or in a screening setting. A limitation of optical imaging in comparison to
PET imaging is the difficulty of deep tissue imaging and of quantification.
Fluorescent dyes in the near-infrared (NIR) region are the most suitable for use
in imaging. The light emitted after excitation is in the region of 650-900 nm. At
this wavelength tissue absorption and scattering is low, tissue penetration is in
the range of 1 to 2 centimeters and tissue auto-fluorescence in not interfering
with dye signal52. The challenge is to develop a fluorescent probe that maintains
its fluorescent properties after conjugation to mAbs. Studies with ICG, the only
FDA-approved NIR dye, showed promising results when used as a single agent53,
however, when the molecule was modified for coupling to a mAb a serious loss
of fluorescence was observed54. Different fluorescent dyes have been tested for
coupling abilities and the dye with the highest potential to be used in the clinical
setting is IRDye800CW (Licor Bioscience). IRDye800CW can be functionalized
with either an NHS or a maleimide reactive group, ideal for conjugation to mAbs,
as illustrated in several preclinical studies with IRDye800CW-mAb conjugates55-59.
The first clinical trials with IRDye800CW labeled mAbs – bevacizumab and cetuximab – are currently ongoing (Clinicaltrials.gov, NCT01987375, NCT02113202,
NCT01508572, NCT01972373, NCT02129933).
Even more attractive seems the use of dual-labeled mAbs, since combining PET
and optical imaging techniques would provide the ideal probe: the location and
size of the tumor and metastases can be determined with immuno-PET, followed
by a complete tumor resection aided by imaging of the NIR-labeled mAb during surgery. For these dual-labeled conjugates with IRDye800CW, FDA-approved
mAbs trastuzumab60 and bevacizumab61 as well as new mAbs were used. Also different PET radionuclides were used. Besides the expected 89Zr62,63, also 64Cu (t1/2=
12.7 hours) was investigated64, 65. All studies show tumor specific uptake of the
conjugate with both imaging modalities, confirming the potential of IRDye800CW.
However, clinical translation of dual-labeled conjugates still has some hurdles to
take. Clinical use of 64Cu with intact mAbs seems less desirable, since the half-life
of 64Cu does not match the biological half-life of intact mAbs in humans. Moreover,
General introduction
stable coupling of 64Cu to mAbs is challenging, and sequestration of 64Cu in the
liver might occur. In addition, the number of conjugated dye and chelator groups
per mAb was different for every study performed, and this might affect antigen
binding and pharmacokinetics. When clear protocols would be available for the
reproducible production of stable dual-labeled mAbs, clinical use of PET/NIR
molecules will most probably be the next translational step.
OUTLINE OF THIS THESIS
As outlined above in Chapter 1, mAbs have shown to be very promising candidates
in cancer therapy as naked single agents, but mostly when used as carrier for tumorspecific delivery of potent toxic compounds. The latter requires a reproducible
synthesis protocol that is relatively straightforward and GMP compliant. Moreover,
labeling of the mAbs and mAb-conjugates facilitates product development and
their clinical applications. The more toxic the naked mAb or the toxic compound
to be delivered by the mAb are, the more important it becomes that tumor targeting
is “precise”; precise at the level of the individual patient and precise at the level
of a high uptake in the tumor and a low uptake in the normal tissues. In this thesis
the development of tools to allow the quantitative tracking of mAbs in vivo by PET
or optical imaging is described, which might be of help in steering efforts in the
development and (pre)clinical application of precision medicine with mAbs and
other biologicals.
In Chapter 2, we focus on the use of radioactive labels in the development and in
vivo validation of novel antibody drug conjugates (ADCs). ADCs comprise a class
of drugs receiving a lot of attention from pharmaceutical companies involved in the
development of anti-cancer drugs (see General introduction). We introduce a new
family of highly potent drugs, tubulysins, to be coupled to mAbs as novel ADCs.
For efficient ADC development, tubulysins are labeled with 131I, while the mAb
under investigation in this study (trastuzumab) is labeled with 89Zr. By using this
approach of “dual-radiolabeling” we monitor the direct conjugation of tubulysin
derivatives to trastuzumab, to get full control over drug-antibody ratio (DAR), and
we characterize the tubulysin-trastuzumab conjugates for their pharmacokinetics
and tumor targeting properties. Finally, we describe the in vivo efficacy of these
ADCs, which have been developed by guidance of radiolabeling technology.
As outlined in the introduction section, 89Zr labeling of mAbs allows the quantitative in vivo characterization of mAbs and other biologicals using PET imaging.
PET imaging is particularly well suited for whole body imaging, but is not qualified
to show tumor targeting at the cellular level. For the latter, labeling of mAbs with
23
1
24
Chapter 1
fluorescent dyes in combination with optical imaging, is better suited as it allows
high resolution and real time imaging. Irrespective the use of PET radionuclides or
fluorescent dyes for mAb tracking, to be acceptable for pharmaceutical companies
and regulatory authorities, the procedures for coupling to mAbs should we well
controlled and inert, not affecting the biological properties of the mAb.
In Chapter 3, we use 89Zr labeling as an internal reference standard to prove that
labeling of the mAbs cetuximab and bevacizumab with the NIR dye IRDye800CW
can be performed in a controlled an inert way, without affecting binding and
biodistribution characteristics of the mAbs. To this end, different equivalents of
IRDye800CW are coupled to the mAbs.
To allow broad scale preclinical and clinical application of single- as well as of
dual-modal fluorescence and PET imaging, Chapter 4 presents a detailed protocol
for the inert coupling of IRDye800CW and/or 89Zr to mAbs in a current good
manufacturing practice-compliant (cGMP) way. The previous study (Chapter 3)
provided the necessary information to design a user and product friendly protocol:
to avoid unnecessary radiation exposure to the product and personnel, first the
chelator desferal (Df) is conjugated to the mAb, second the NIR-dye IRDye800CW
is coupled, and finally the conjugate is labeled with 89Zr.
While imaging of mAbs labeled with NIR dyes showed promising results, quantification of the fluorescent signal is more challenging. In Chapter 5, we evaluate
a new method to quantify the uptake of IRDye800CW-cetuximab in organs and
tumors. Also here we use dual-labeling with 89Zr and standard gamma ray quantification method as reference and for validation purposes.
In Chapter 6 a summary of the results is given as well as a general discussion and
future perspectives on the topics. In Chapter 7 a summary in Dutch is provided.
General introduction
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Hericourt J, Richet CH. De la serotherapie dans le traitement du cancer. CR Hebd Acad Sci
1895;120:567-9.
Ehrlich P. Experimental researches on specific therapy on immunity with special reference to the
relationship between distribution and action of antigens. Collected papers 1908;3:1956-60.
Porter RR. The structure of antibodies. The basic pattern of the principal class of molecules that
neutralize antigens (foreign substances in the body) is four cross-linked chains. This pattern is
modified so that antibodies can fit different antigens. Sci Am 1967;217:81-7 passim.
Edelman GM, Gall WE, Waxdal MJ, Konigsberg WH. The covalent structure of a human gamma
G-immunoglobulin I. Isolation and characterization of the whole molecule, the polypeptide
chains, and the tryptic fragments. Biochemistry 1968;7:1950-8.
Kunkel HG, Slater RJ, Good RA. Relation between certain myeloma proteins and normal gamma
globulin. Proc Soc Exp Biol Med 1951;76:190-3.
Potter M, Boyce CR. Induction of plasma-cell neoplasms in strain BALB/c mice with mineral oil
and mineral oil adjuvants. Nature 1962;193:1086-7.
Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7.
Green C, Murray L, Hortobagyi N. Monoclonal antibody therapy for solid tumors. Cancer Treatment Reviews 2000;26:269-86.
van Dongen GA, Vosjan MJ. Immuno-Positron Emission Tomography: shedding light on clinical
antibody therapy. Cancer Biother Radiopharm 2010;25:375-85.
Stern M, Herrmann R. Overview of monoclonal antibodies in cancer therapy: present and
promise. Critical Reviews in Oncology/Hematology 2005;54:11-29.
Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with
monoclonal antibodies. Blood 1998;91:1644-52.
Scolnik PA. mAbs: A business perspective. MAbs 2009;1:179-84.
Hudis CA. Trastuzumab--mechanism of action and use in clinical practice. N Engl J Med 2007;
357:39-51.
Mukherji SK. Bevacizumab (Avastin). AJNR Am J Neuroradiol 2010;31:235-6.
Price TJ, Peeters M, Kim TW, Li J, Cascinu S, Ruff P, et al. Panitumumab versus cetuximab in
patients with chemotherapy-refractory wild-type KRAS exon 2 metastatic colorectal cancer
(ASPECCT): a randomised, multicentre, open-label, non-inferiority phase 3 study. Lancet Oncol
2014;15:569-79.
Barth MJ, Mavis C, Czuczman MS, Hernandez-Ilizaliturri FJ. Ofatumumab exhibits enhanced in
vitro and in vivo activity compared to rituximab in pre-clinical models of mantle cell lymphoma.
Clin Cancer Res 2015;21:.
Czuczman MS, Fayad L, Delwail V, Cartron G, Jacobsen E, Kuliczkowski K, et al. Ofatumumab
monotherapy in rituximab-refractory follicular lymphoma: results from a multicenter study.
Blood 2012;119:3698-704.
Glassman PM, Balthasar JP. Mechanistic considerations for the use of monoclonal antibodies for
cancer therapy. Cancer Biol Med 2014;11:20-33.
Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin
Cancer Res 2011;17:6958-62.
25
1
26
Chapter 1
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Scheuer W, Friess T, Burtscher H, Bossenmaier B, Endl J, Hasmann M. Strongly enhanced antitumor activity of trastuzumab and pertuzumab combination treatment on HER2-positive human
xenograft tumor models. Cancer Res 2009;69:9330-6.
Baselga J, Cortés J, Kim SB, Im SA, Hegg R, Im YH, et al. Pertuzumab plus trastuzumab plus
docetaxel for metastatic breast cancer. N Engl J Med 2012;366:109-19.
Karagiannis TC. Comparison of different classes of radionuclides for potential use in radioimmunotherapy. Hell J Nucl Med 2007;10:82-8.
Schrama D, Reisfeld RA, Becker JC. Antibody targeted drugs as cancer therapeutics. Nat Rev
Drug Discov 2006;5:147-59.
Redman JM, Hill EM, AlDeghaither D, Weiner LM. Mechanisms of action of therapeutic antibodies for cancer. Molecular Immunology 2015;67:28-45.
Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer drug
conjugates. Bioorg Med Chem 2005;13:5043-54.
McCombs JR, Owen SC. Antibody drug conjugates: design and selection of linker, payload and
conjugation chemistry. AAPS J 2015;17:339-51.
Lambert JM. Drug-conjugated monoclonal antibodies for the treatment of cancer. Curr Opin
Pharmacol 2005;5:543-9.
Gerber HP, Koehn FE, Abraham RT. The antibody-drug conjugate: an enabling modality for
natural product-based cancer therapeutics. Nat Prod Rep 2013;30:625-39.
Ricart AD. Antibody-drug conjugates of calicheamicin derivative: gemtuzumab ozogamicin and
inotuzumab ozogamicin. Clin Cancer Res 2011;17:6417-27.
Bornstein GG. Antibody drug conjugates: preclinical considerations. AAPS J 2015;17:525-534.
Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al. Targeting HER2positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer
Res 2008;68:9280-90.
Amiri-Kordestani L, Blumenthal GM, Xu QC, Zhang L, Tang SW, Ha L, et al. FDA approval:
ado-trastuzumab emtansine for the treatment of patients with HER2-positive metastatic breast
cancer. Clin Cancer Res 2014;20:4436-41.
Shor B, Gerber HP, Sapra P. Preclinical and clinical development of inotuzumab-ozogamicin in
hematological malignancies. Mol Immunol 2015;67:107-16.
Li D, Poon KA, Yu SF, Dere R, Go M, Lau J, et al. DCDT2980S, an anti-CD22-monomethyl
auristatin E antibody-drug conjugate, is a potential treatment for non-Hodgkin lymphoma. Mol
Cancer Ther 2013;12:1255-65.
Mullard A. Maturing antibody-drug conjugate pipeline hits 30. Nat Rev Drug Discov 2013;12:
329-32.
Ott PA, Hamid O, Pavlick AC, Kluger H, Kim KB, Boasberg PD, et al. Phase I/II study of the
antibody-drug conjugate glembatumumab vedotin in patients with advanced melanoma. J Clin
Oncol 2014;32:3659-66.
Bendell J, Saleh M, Rose AA, Siegel PM, Hart L, Sirpal S, et al. Phase I/II study of the antibodydrug conjugate glembatumumab vedotin in patients with locally advanced or metastatic breast
cancer. J Clin Oncol 2014;32:3619-25.
Berdeja JG. Lorvotuzumab mertansine: antibody-drug-conjugate for CD56+ multiple myeloma.
Front Biosci (Landmark Ed) 2014;19:163-70.
Whiteman KR, Johnson HA, Mayo MF, Audette CA, Carrigan CN, LaBelle A, et al. Lorvotuzumab
mertansine, a CD56-targeting antibody-drug conjugate with potent antitumor activity against
small cell lung cancer in human xenograft models. MAbs 2014;6:556-66.
General introduction
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
Evans JB, Syed BA. From the analyst’s couch: Next-generation antibodies. Nat Rev Drug Discov
2014;13:413-4.
Shuptrine CW, Surana R, Weiner LM. Monoclonal antibodies for the treatment of cancer. Semin
Canc Biol 2012;22:3-13.
Postow MA, Callahan MK, Wolchok JD. Immune checkpoint blockade in cancer therapy. J Clin
Oncol 2015;33:1974-82.
Sliwkowski MX, Mellman I. Antibody therapeutics in cancer. Science 2013;341:1192-8.
AlDeghaither D, Smaglo BG, Weiner LM. Beyond peptides and mAbs--current status and future
perspectives for biotherapeutics with novel constructs. J Clin Pharmacol 2015;55 Suppl 3:S4-20.
Wu AM, Olafsen T. Antibodies for molecular imaging of cancer. Cancer J 2008;14:191-7.
Verel I, Visser GW, Boerman OC, van Eerd JE, Finn R, Boellaard R, et al. Long-lived positron
emitters zirconium-89 and iodine-124 for scouting of therapeutic radioimmunoconjugates with
PET. Cancer Biother Radiopharm 2003;18:655-61.
Vosjan MJ, Perk LR, Visser GW, Budde M, Jurek P, Kiefer GE, et al. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate
p-isothiocyanatobenzyl-desferrioxamine. Nat Protoc 2010;5:739-43.
Nayak TK, Brechbiel MW. Radioimmunoimaging with longer-lived positron-emitting radionuclides: potentials and challenges. Bioconjug Chem 2009;20:825-41.
Boerman OC, Oyen WJ. Immuno-PET of cancer: a revival of antibody imaging. J Nucl Med
2011;52:1171-2.
Knowles SM, Wu AM. Advances in immuno-positron emission tomography: antibodies for
molecular imaging in oncology. J Clin Oncol 2012;30:3884-92.
Wright BD, Lapi SE. Designing the magic bullet? The advancement of immuno-PET into clinical
use. J Nucl Med 2013;54:1171-4.
Bai M, Bornhop DJ. Recent advances in receptor-targeted fluorescent probes for in vivo cancer
imaging. Curr Med Chem 2012;19:4742-58.
Polom K, Murawa D, Rho YS, Nowaczyk P, Hünerbein M, Murawa P. Current trends and emerging future of indocyanine green usage in surgery and oncology: a literature review. Cancer 2011;
117:4812-22.
Ito S, Muguruma N, Hayashi S, Taoka S, Bando T, Inayama K, et al. Development of agents for
reinforcement of fluorescence on near-infrared ray excitation for immunohistological staining.
Bioorg Med Chem 1998;6:613-8.
Yang Y, Zhang Y, Hong H, Liu G, Leigh BR, Cai W. In vivo near-infrared fluorescence imaging of
CD105 expression during tumor angiogenesis. Eur J Nucl Med Mol Imaging 2011;38:2066-76.
Vermeulen JF, van Brussel AS, Adams A, Mali WP, van der Wall E, van Diest PJ, et al. Nearinfrared fluorescence molecular imaging of ductal carcinoma in situ with CD44v6-specific
antibodies in mice: a preclinical study. Mol Imaging Biol 2013;15:290-8.
Day KE, Sweeny L, Kulbersh B, Zinn KR, Rosenthal EL. Preclinical comparison of near-infraredlabeled cetuximab and panitumumab for optical imaging of head and neck squamous cell
carcinoma. Mol Imaging Biol 2013;15:722-9.
Bhattacharyya S, Patel N, Wei L, Riffle LA, Kalen JD, Hill GC, et al. Synthesis and biological
evaluation of panitumumab-IRDye800 conjugate as a fluorescence imaging probe for EGFRexpressing cancers. Med Chem Commun 2014;5:1337-46.
Korb ML, Hartman YE, Kovar J, Zinn KR, Bland KI, Rosenthal EL. Use of monoclonal antibodyIRDye800CW bioconjugates in the resection of breast cancer. J Surg Res 2014;188:119-28.
27
1
28
Chapter 1
60.
61.
62.
63.
64.
65.
Sampath L, Kwon S, Hall MA, Price RE, Sevick-Muraca EM. Detection of cancer metastases with
a dual-labeled near-infrared/positron emission tomography imaging agent. Transl Oncol 2010;
3:307-217.
Zhang Y, Hong H, Engle JW, Yang Y, Barnhart TE, Cai W. Positron emission tomography and
near-infrared fluorescence imaging of vascular endothelial growth factor with dual-labeled
bevacizumab. Am J Nucl Med Mol Imaging 2012;2:1-13.
Zhang Y, Hong H, Severin GW, Engle JW, Yang Y, Goel S, et al. ImmunoPET and near-infrared
fluorescence imaging of CD105 expression using a monoclonal antibody dual-labeled with (89)
Zr and IRDye 800CW. Am J Transl Res 2012;4:333-46.
Hong H, Zhang Y, Severin GW, Yang Y, Engle JW, Niu G, et al. Multimodality imaging of breast
cancer experimental lung metastasis with bioluminescence and a monoclonal antibody duallabeled with 89Zr and IRDye 800CW. Mol Pharm 2012;9:2339-49.
Zhang Y, Hong H, Orbay H, Valdovinos HF, Nayak TR, Theuer CP, et al. PET imaging of CD105/
endoglin expression with a 61/64Cu-labeled Fab antibody fragment. Eur J Nucl Med Mol Imaging
2013;40:759-67.
Hall MA, Pinkston KL, Wilganowski N, Robinson H, Ghosh P, Azhdarinia A, et al. Comparison
of mAbs targeting epithelial cell adhesion molecule for the detection of prostate cancer lymph
node metastases with multimodal contrast agents: quantitative small-animal PET/CT and NIRF. J
Nucl Med 2012;53:1427-37.