Bevacizumab Improves Survival for Metastatic Colorectal Cancer Patients
1. The addition of bevacizumab to oxaliplatin, fluorouracil, and leucovorin
improves survival duration for patients with previously treated metastatic
colorectal cancer.
A Bevacizumab “Success” Story
J. Clin. Onc. (2007) 25, 1539-1544
829 patients, metastatic colon cancer, treated with standard therapy or
standard therapy + bevacizumab
The median duration of survival for the group treated with FOLFOX4 and
bevacizumab was 12.9 months compared with 10.8 months for the group
treated with FOLFOX4 alone (corresponding hazard ratio for death 0.75; P
.0011), and 10.2 months for those treated with bevacizumab alone.
1 year survival SC+B, 56%; SC, 43%
2. Biological Agents Targeting the Same Molecule
Chimeric Antibody Fully Human Antibody
Soluble TNF receptor,
normally involved in
down regulating
inflammatory responses
Fc region only present
for stability in serum
Intercept: obstruct (someone or
something) so as to prevent
them from continuing to a
destination
3. So what is Etanercept Intercepting?
TNF, tumor necrosis factor
• Name derived from an experiment performed in 1896
demonstrating that bacterial extracts could shrink tumors
• Extracts contained bacterial lipopolysaccharide which
stimulated inflammation and induced TNF
4. Goal: Develop Drugs to Treat Chronic
Inflammatory Diseases
Rheumatoid Arthritis
• ~ 1% of population
• average age of onset 40 years
• female/male 3:1
• joint erosion within 2 years of
diagnosis, disabilities after 5 years
• average medical costs $23,00/year
• chronic pain, stiffness of joints;
tiredness and lethargy
5. Tumor Necrosis Factor (TNF): A Cytokine Target for
Treating Rheumatoid Arthritis
Anti-TNF
Early studies suggested TNF might be a master regulator of the inflammatory process
6. Encouraging Data from First Phase I Trail for Infliximab
CRP: C reactive protein, a marker for
inflammation
7. Phase II Trial Indicates Infliximab Synergizes with
Methotrexate
Loss of response correlates with
patient antibodies to Infliximab
Immunosuppressive effects of
methotrexate likely reduce
patients ability to raise antibodies
to Infleximab
HUMA: human anti-mouse antibodies
In the setting of Crohn’s disease ~ 60% of patients raise antibodies to
Infliximab by the 5th injection
8. Crohn’s Disease and Ulcerative Colitis are Other
Chronic Inflammatory Diseases Where Anti-TNF
Therapy is Indicated
9. HUMA Responses to Different Anti-TNF Drugs
Infliximab – 51, 21, and 7% of patients raised antibodies in response to 1, 3,
and 10 mg/kg dosing
Fewer antibodies and higher doses of drug suggest tolerance mechanisms
may come into play
Adalimumab – less immunogenic 6% of patients raised antibodies at the
lowest dose
Etanercept – less immunogenic 2% of patients raised antibodies at the
standard dose
Antibodies will always have novel sequences in their antigen binding regions –
antibodies against these regions are called anti-idiotype antibodies
10. Success in Treating Chronic Inflammatory Diseases:
At What Cost?
Serious infections have happened in people taking HUMIRA. These serious infections
include tuberculosis (TB) and infections caused by viruses, fungi, or bacteria that have
spread throughout the body. Some people have died from these infections. Your doctor
should test you for TB before starting HUMIRA, and check you closely for signs and
symptoms of TB during treatment with HUMIRA. If your doctor feels you are at risk, you
may be treated with medicine for TB.
Cancer. For children and adults taking TNF blockers, including HUMIRA, the chance of
getting lymphoma or other cancers may increase. There have been cases of unusual
cancers in children, teenagers, and young adults using TNF blockers. Some people have
developed a rare type of cancer called hepatosplenic T-cell lymphoma. This type of
cancer often results in death. If using TNF blockers including HUMIRA, your chance of
getting two types of skin cancer (basal cell and squamous cell) may increase. These
types are generally not life-threatening if treated; tell your doctor if you have a bump or
open sore that doesn’t heal.
11. Approaches to Isolation of Fully Human Antibodies
(Adalimumab): Phage Display
Each individual phage particle embodies
both phenotype (expressed antibody)
and genotype (the gene encoding the
antibody)
Select for a phage particle that binds to a
particular antigen and you immediately
have the gene encoding the antibody
responsible for binding
Fusion protein produced between
antibody fragment and gene 3 protein
17. Fully Human Antibodies: Transgenic Mice
• Human heavy (1.3 mg) and κ light
chain (1.8 mb) on chromosomes 14
and 2, respectively
• Heavy: 95 V, 30 D, 6 J, and the
constant region
• Light: 76 V, 65J, and the constant
region
18. Expression of Human Immunoglobulins by
Xenomouse (II) Mice
B lymphocyte
marker
wild-type mice knockout mice Xenomouse (II) mice
19. Xenomouse (II) is Capable of Producing High Affinity
Fully Human Antibodies
20. Is Ofatumumab Better than Rituximab?
Ofatumumab is a fully human antibody produced in transgenic mice
immunized with CD20
• UK-based GlaxoSmithKline and Danish biotech firm Genmab have reported
results from a Phase III trial (ORCHARRD) of ofatumumab (Arzerra) plus
chemotherapy versus rituximab plus chemotherapy to treat patients with
relapsed or refractory diffuse large B-cell lymphoma (DLBCL)
• The trial did not meet its primary endpoint as there was no statistically significant
difference in progression free survival (PFS) between the treatment arms.
• According to the firms, there were no differences in adverse events (AEs) leading
to treatment discontinuation, Grade >3 AEs, severe adverse events (SAEs), or
fatal SAEs between the treatment arms.
• "Based on today's results, we are unlikely to move forward with a regulatory
filing," Winkel said.
21. Allora, Is There Anything Our There that can Beat
Rituximab in a Head to Head Trial?
Enter Obinutuzumab, a glyco-engineered humanized antibody
• Virtually identical Kd to CD20 as rituximab
• Type I antibodies cluster CD20 in
lipid rafts and show a bias toward
CDCC
• Type II antibodies show a bias
toward more direct effects
Differences reside in mechanisms of
target destruction
22. Antibodies are Glycoproteins and Glycosylation can
Influence Effector Function
Antibodies are subject to N-
linked glycosylation at residue
297 of the Fc region
23. Glycoproteins
N-linked glycosylation – oligosaccharides linked to Asn residues
in the sequence context
Asn-X-Ser or Asn-X-Thr
Proteins are initially modified by a 14 residues core oligosaccharide
Dolichol is a lipid
CH2
C ON
Sugar
N-glycosidic bond
H
unusual high energy phosphate
ester linkage provides energy for
forming N-glycosidic bond
24. Cotranslational Addition of the Core Oligosaccharide
oligosaccharide transferase
targeting
signal
cytoplasm
rough ER
membrane
ER lumen
complex
high mannose
Asn-X-Ser
Asn-X-Thr
25. Processing the Core Oligosaccharide
Glucose
Mannose
N-acetyl
glucosamine
Fucose
Galactose
Sialic acid
High mannose
class switches
from trimming to
addition
Complex
class
switches
from
trimming to
addition
26. Cell Death Assays: Annexin Staining
Phosphatidylserine (PS) normally found on cytoplasmic surface of plasma
membrane
During intermediate stages of apoptosis PS is translocated to the outer surface of
the plasma membrane
Externalization of PS makes it available for binding to fluorescent PS-binding
proteins (Annexins)
Editor's Notes
Etanercept is a fusion protein produced by recombinant DNA. It fuses the TNF receptor to the constant end of the IgG1 antibody. First, the developers isolated the DNA sequence that codes the human gene for soluble TNF receptor 2, which is a receptor that binds to tumor necrosis factor-alpha. Second, they isolated the DNA sequence that codes the human gene for the Fc end of immunoglobulin G1 (IgG1). Third, they linked the DNA for TNF receptor 2 to the DNA for IgG1 Fc. Finally, they expressed the linked DNA to produce a protein that links the protein for TNF receptor 2 to the protein for IgG1 Fc.