Monoclonal Antibodies as a novel class of cancer therapeutics

UNIVERSITÀ DEGLI STUDI DI BRESCIA
Monoclonal Antibodies as
a novel class of
cancer therapeutics
Patrizia Alessi
07.01.2008
The concept of "magic bullets" for the treatment of disease
site of disease (e.g., tumor)
What are antibodies?
Antibodies are immune system-related proteins called immunoglobulins
produced by B lymphocyte/plasma cells in response to the presence in the
body of antigens: (i.e., foreign proteins or polysaccharides derived form
bacteria, toxins, yeast, viruses or other cells).
Antibodies (or immunoglobulins ) generally assume one of two roles:
1) plasma membrane bound antigen receptors on the surface of a B-cell
2) free antibodies in cellular fluids functioning to intercept and eliminate
antigenic determinants.
Antibody structure
Immunoglobulins are composed of four polypeptide chains: two "light" chains
and two "heavy" chains.
"Hypervariable" regions, or "Complementarity Determining Regions" (CDRs)
are found within the variable regions of both the heavy and light chains.
These regions serve to recognize and bind specifically to antigen.
Antibody type
The type of heavy chain determines the immunoglobulin isotype (IgA, IgD,
IgG, IgE, IgM, respectively). Each chain has "constant" and "variable" regions
as shown in the figure. Variable regions are contained within the amino (NH2)
terminal end of the polypeptide chain (amino acids 1-110).
Antibody: different parts with different functions
Fab & Fc
IgG
Monoclonal antibody: classical procedure
Hybridoma technique
Hybridoma technique
Pros
- Reliable technique taking advantage of an “in vivo affinity maturation
process” crucial for the generation of high affinity MAb.
Cons
-If the antigen is identical or very homologous between the rodent
species and humans, such "anti-self" antibodies are difficult or
impossible to obtain.
Rodent immunoglobulins have sequences that are recognized as
strong antigens by the human immune system, thus preventing
repeated administration in vivo in humans (i.e., HAMA reactions:
Human anti-Mouse antibody).
Chimeric or Humanized MAb are thus necessary for clincal use.
- Time consuming
Monoclonal antibody: NEW TECHNIQUES (1)
One promising way to increase antibody yield or develop new antibodies
may be by using genetically altered animals.
Abgenix, a company in Fremont, CA, has developed the transgenic
“XenoMouse” in which the mouse antibody-producing genes have been
inactivated and functionally replaced by approximately 90% of the human Ig
gene loci in germline configuration, coding for the heavy and [kappa] light
chains.
Upon immunization with any specific human or nonhuman antigens, the
“XenoMouse” generates MAbs, which are fully human Igs, with high
affinities and antigen-binding specificities.
“XenoMouse” strains producing specifically IgG1, IgG2, or IgG4 isotypes
have also been created to generate panels of diverse and highly specific
MAbs.
Monoclonal antibody: NEW TECHNIQUES
Kirin Brewery Company, Japan, has developed another transgenic mouse
known as the “Trans-Chromo” mouse.
The endogenous IgH and IgG loci of the “Trans-Chromo” mouse were
inactivated, but it harbors 2 individual human chromosome fragments,
derived from human chromosomes 2 and 14, that contain whole human Ig
light- and heavy-chain loci, respectively.
These mice are capable of producing every subtype of fully human Ig,
including IgA and IgM.
In these transgenic mouse models, human antibodies with high affinity to
an immunized antigen are naturally selected by the murine immune system
via an affinity maturation process, and thereby show increased diversity of
the MAbs.
Monoclonal antibody: NEW TECHNIQUES (1)
At present at least 33 human Ab produced in transgenic mice are in clinical
development, including 5 in Phase III clinical trials:
1)
panitumumab Abgenix, Inc.and Amgen Inc., an epidermal-grow-factor
receptor (EGFR) specific MAb for the treatment of colorectal cancer
2)
ipilimumab Medarex, Inc. and Brystol-Myers Squibb Company, a
cytotoxic T-lynphocyte antigen-4 (CTLA-4) for the treatment of melanoma
3)
denosumab Amgen Inc. a receptor activator of nuclear factor-κB (RANK)
ligand-specific MAb for post-menopausal osteoporosis and to avoid
bone loss in hormone-ablation therapy during treatment of cancer
4)
zanolimumab (HuMax-CD4) Genmab A/S and Serono S.A. a CD4 specific Ab
for treatment of cutaneous T-cell lymphoma
5)
golimumab Centocor, Inc a tumor necrosis factor (TNF) specific MAb for
treatment of rheumatoid arthritis, psoriatic arthritis and ankylosing
spondylitis.
Transgenic mouse
Pros:
Fully human antibodies with high affinity to an immunized antigen.
(i.e., are naturally selected by the immune system via an affinity maturation
process, and thereby show increased diversity of the MAbs).
Cons:
- If the antigen is a self protein for humans, such "anti-self" antibodies are
difficult to obtain.
- More time consuming (immune response is less robust in transgenic
animal)
- Difficult to obtain species/crossreactive Ig
- Technology nor available to the majority of Lab worldwide.
- Expensive technology
Monoclonal antibody: NEW TECHNIQUES (2)
Antibody Phage Display
One human antibody isolated by phage-display has been approved widely
for therapeutic use: adalimumab (Humira) Abbott Lab a TNF-specific MAb
used for treatment of rheumatoid and psoriatic arthritis
Monoclonal Antibodies
as Novel anti-Cancer Therapies
Therapeutic antibody
Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated
cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.
MAbs for cancer immunotherapy
Since the mid-1990, antibodies have emerged as an important new drug
class. Indeed, 18 antibodies are now approved (17 have been marketed and
1 withdrawn) for therapeutic use in the United States across diverse clinical
setting, including oncology, chronic inflammatory diseases, transplantation,
infectious diseases and cardiovascular medicine.
These approved antibody therapeutics include 14 unmodified IgG,
1antibody-drug conjugate and 1 Fab.
At least 150 antibodies are in clinical development.
Some of these are chimeric or humanized antibodies developed in the early
days of antibody engineering, whereas others are derived from novel
technologies.
Bioparmaceutical in the pipeline 2006
Potent MAb therapeutics by design
Abs belong to a well-established drug class that has a high success rate
from the first use in humans to regulatory approval: 29% for chimeric Abs,
25% for humanized Abs (only 11% small-molecule drug).
Abs are generally well tolerated by humans, although infusion reaction
(first dose) are common but manageable (e.g.: rituximab).
Their clinical potential can readily be increased by improving their existing
activity or by endowing them with new activities.
Restriction of the targets of those on the surface or exterior of host cells or
Invading pathogens. For the antigen, factors that determine the efficacy of the
targeting of the antibody include specific tissue expression, abundance,
availability, number of antigens per cell, and antigen (receptor) shedding.
Antibody drugs are expensive, usage limitation.
Adverse events (natalizumab, Tysabri)
Iterative design of antibody therapeutics
Goals for antibody therapeutics
Improve efficacy = improve antitumor activity and patient survival
(combination therapy)
Improve safety = increasing the potency and the half life in plasma
(dose or frequency of administrator can be reduced)
Reduce cost = use of alternative host for production
FDA approved MAb for cancer immunotherapy
Muromonab-CD3
Johnson & Johnson New Brunswick,
New Jersey
Orthoclone
OKT3
Murine, IgG2a, anti-CD3
Rituximab
Genentech
Rituxan
Chimeric, IgG1 , anti-CD20
Daclizumab
Hoffmann-La Roche Basel
Zenapax
Humanized, IgG1 , anti-CD25
Basiliximab
Novartis Basel
Simulect
Chimeric, IgG1 , anti-CD25
Palivizumab
MedImmune Gaithersburg, Maryland
Synagis
Humanized, IgG1 , anti-respiratory syncytial
virus
Infliximab
Centocor
Remicade
Chimeric, IgG1 , anti-tumor necrosis factor
(TNF )
Trastuzumab
Genentech
Herceptin
Humanized, IgG1 , anti-HER2
Gemtuzumab
ozogamicin
Wyeth Madison, New Jersey
Mylotarg
Humanized, IgG4 , anti-CD33; immunotoxin
Alemtuzumab
Genzyme Cambridge, Massachusetts
Campath-1H
Humanized, IgG1 , anti-CD52
Ibritumomab
tiuxetan
Biogen Idec
Zevalin
Murine, IgG1 , anti-CD20; radiolabeled (Yttrium
90)
Adalimumab
Abbott Deerfield Park, Illinois
Humira
Human, IgG1 , anti-TNF
Tositumomab-I131
Corixa Seattle
Bexxar
Murine, IgG2a , anti-CD20; radiolabeled (Iodine
131)
Cetuximab
Imclone Systems New York
Erbitux
Chimeric, IgG1 , anti-Epidermal growth
factor receptor
Bevacizumab
Genentech
Avastin
Humanized, IgG1, anti-vascular endothelial
growth factor
Tunable properties of antibodies
Immunigenicity is influenced by biochemical (sequence, origin, stability,
modification), clinical parameter (dose, route and frequency of
administration) and patient-specific factors (disease and immune status,
MHC haplo-type and concomitant medication.
Immunogenicity
Hybridoma technique
3% success
Poor effector functions
Poor half life in plasma
MAbs therapeutics generation
Tunable properties of antibodies
Immunigenicity is influenced by biochemical (sequence, origin, stability,
modification), clinical parameter (dose, route and frequency of
administration) and patient-specific factors (disease and immune status,
MHC haplo-type and concomitant medication.
Antigen binding specificity: cross-species reactivity for evaluation of
efficacy and toxicity.
Antigen binding-affinity: increasing Kd (display technologies)
Biological activities associated with variable domains : increasing
efficacy and biological potency by functional screening after affinity
maturation, correct selection of the Ab format.
Antibody formats
How to improve performances
Effector functions: the right isotype
Intact antibodies targeted to bacteria, viruses or cells can mediate target
elimination through the interaction of macrophages, neutrophils and
natural killer cells, which induce phagocytosis and antibody-dependent
cellular cytotoxicity (ADCC), as well as through activation of the
complement system (CDC).
Because the interaction between antibodies and effector molecules, such
as Fc receptors or complement, is mediated by the Fc part of the
antibody molecule, antibody fragments without Fc cannot attract such
effectors.
A prerequisite for effective target elimination comprises crucial elements
both within the antigen and the antibody
Antibody effector functions
Tunable properties of antibodies
Pharmacokinetics: the terminal half life of antibodies in plasma can be
tuned over a wide range to fit clinical goals. A way is to tailor the
interaction Fc and FcRn. Another is the PEGylation…
Molecular architecture: the modular domain architecture
or
No Fc effector functions
PEGylation
More efficient penetration of tumor
Faster clearance
Greater tumor uptake
Binding without crosslinking (monovalent)
Small size
High affinity, solubility and robust expression
Camels, Llamas and sharks
?
Mode of actions for Mabs 1980-2005
Tunable properties of antibodies
Internalization is desirable for certain applications, such as for the
delivery of cytotoxic drug and immunoliposomes
Biophysical properties such as stability, solubility and folding kinetics
(role of the framework)
Cancer Immunotherapy with MAb
Examples of Monoclonal antibody endowed with therapeutic efficacy against
human tumors:
ERBITUX® (Cetuximab) = Anti-EGF-r (EU 2004)
Herceptin (Trastuzumab) = Anti-HER2/neu (EU 2000)
Avastin (bevacizumab) = Anti-VEGF (EU 2005)
HER2/neu and Cancer
•
The human HER-2/neu (c-erbB2) gene product, like the epidermal
growth factor receptor, is a transmembrane receptor protein that includes
an intracellular tyrosine kinase domain.
•
In normal cells, two copies of the gene per cell (one on each
chromosome 17) are present. However, in the presence of HER-2/neu
amplification there may be as many as 50 or 100 HER-2/neu genes per
tumor cell. This gene amplification results in overexpression of p185
HER-2/neu at both the mRNA and protein levels. As a result, instead of ~
20,000 to 50,000 HER-2/neu molecules per cell, there can be as many
as 2,000,000 molecules per cell in neoplastic tissues.
•
HER2/neu overexpression has been previously suggested to represent a
major prognostic factor in several human cancers including endometrial,
breast and ovarian carcinomas and to be correlated with resistance to
chemotherapy
HER2/neu and Breast Cancer
•
An estimated 211,240 women will be diagnosed with breast cancer in the
United States in 2005. Of these, about 30 percent have lymph node-positive
breast cancer, and about 20 percent of these tumors overexpress the HER2 protein, the target for trastuzumab.
•
Breast cancer is the most commonly diagnosed cancer in women and the
second leading cause of cancer-related death in women in this country. An
estimated 40,110 deaths from female breast cancer will occur in 2005 in the
United States, accounting for about 15 percent of all cancer-related deaths
in women in the nation.
What is Herceptin ?
Herceptin® (Trastuzumab) is the only FDA-approved therapeutic for
HER2 protein overexpression metastatic breast cancer.
Herceptin is a therapy for women with metastatic breast cancer
whose tumors have too much HER2 protein.
For patients with this disease, Herceptin is approved for first-line
use in combination with paclitaxel, and as a single agent for those
who have received one or more chemotherapy regimens.
Lowering immunogenicity
Technologies for antibody optimization
Construction of humanized gene.
humAb4D5 variants
In vitro potency
Inhibition of SK-BR-3 (breast carcinoma cells) proliferation
In vivo efficacy
In vitro cytotoxicity in combination with paclitaxel
In vivo therapeutic properties
Dose finding
Combination therapy
Data of complete tumor regression
Synergistic interaction at clinically relevant [drug]
Combination therapy
How does Herceptin work
Frequency of efficacy end point events in the Herceptin® Adjuvant (HERA) trial
Is Herceptin effective against Human cancer?
Results from two large randomized clinical trials for patients with HER-2 positive
invasive breast cancer show that those patients with early-stage breast cancer who
received Herceptin® (trastuzumab) in combination with chemotherapy had a significant
decrease in risk for breast cancer recurrence compared with patients who received the
same chemotherapy without trastuzumab.
Patients in the clinical trials who received trastuzumab in combination with standard
combination chemotherapy had a 52 percent decrease in disease recurrence compared
to patients treated with chemotherapy alone. This difference is highly statistically
significant. Among the specific findings from the study were:
At a median follow-up of two years, there were 261 events (such as return of the cancer,
second primary cancer, or death before recurrence) in the control group and 133 events
in the trastuzumab group.
The percentage of patients alive and disease-free at three years was 87.1 percent in the
trastuzumab group and 75.4 percent in the control group. After four years, the respective
percentages were 85.3 percent for trastuzumab vs. 67.1 percent for those on standard
chemotherapy.
Women taking trastuzumab with a particular chemotherapy regimen had a 33 percent
reduction in risk of death (October 20, 2005 New England Journal of Medicine).
From the trastuzumab era to new target therapies: beyond revolution
In the October 2005 edition of The New England Journal of
Medicine, Gabriel Hortobagy claimed that ‘‘the results of
trastuzumab adjuvant trials are not evolutionary but
revolutionary’’
It is obvious that there are certain limitations in the use of this extraordinary drug.
The first is that trastuzumab can only be used in a small number of patients; the
majority (HER-2 negative) cannot benefit from its use.
The second problem is the potential cardiotoxicity of this treatment.
The third dilemma is whether the duration of therapy should be short or
prolonged.
The fourth problem concerns the high cost of trastuzumab treatment to the
National Health Service, which is not to be disregarded.
Acquired and de novo resistance to trastuzumab is a significant clinical
challenge.
Biopharmaceutical benchmarks 2006
What is fueling the biotech engine?
Trends in US sales of MAb
Top companies
Background
Nearly a century ago, Paul Ehrlich first espoused the concept of "magic bullets" for
the treatment of disease. By exploiting inherent differences between healthy and
diseased cells, therapeutic agents could be designed to specifically target diseased
cells while leaving healthy cells unperturbed.
While traditional therapeutic modalities, such as chemotoxic drugs or ionizing
radiation, are very effective at killing tumor cells, they often fail to adequately
discriminate between normal and malignant tissues. Thus, these treatments exact a
heavy toll on the patient and can severely limit the dose or duration of treatment to
suboptimal levels, resulting in poor outcomes for the management of disease, quality
of life, and overall survival.
Clearly, elucidating novel therapeutic strategies to target cytotoxicity exclusively to
malignant cells would greatly reduce deleterious side effects to normal tissues,
enhance the tolerance of the patient to therapy, and result in more effective
treatment.
Christiansen JJ et al., Molecular Cancer Therapeutics, 3(11):1493-501, 2004.
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