therapeutic antibodies 4_humanization (2)
Post on 12-Aug-2015
30 Views
Preview:
TRANSCRIPT
HIF Activation by Hypoxia
histone acetyltransferase
activity
VEGF-A
Epo
Von-Hippel-Lindau tumor suppressor
• tumors require an oxygen supply – biallelic inactivation of VHL leads to a variety of tumors
• congenital mutations in VHL may also lead to polythycemia
cis-acting sequence – hypoxia response element
prolyl hydroxylaseE3 ubiquitin
ligase
erythropoietin a cytokine that promotes red cell production
under hypoxic conditions PHD has reduced activity
Vascular Endothelial Growth Factor (VEGF) Family
• 7 separate genes encoding VEGF family members – VEGF (A-F) and placental growth factor
• splice variants produced from individual genes further increasing diversity
• VEGF-A a prototype
• 165 amino acids• multiple isoforms expressed in different tissues
6 amino acids found at the C-terminus coming
from exon 8a or 8b determines whether factor is pro- or anti-
angiogenic
choice between exons
Differential Splicing Gives Rise to Pro- and Anti-Angiogenic Forms of VEGF-A
favored downstream of HIF activation
3’ proximal splice site
3’ distal splice site
basal expression or promoted by a signaling pathway opposing angiogenesis
exon 7
VEGF-A Structure
ER secretory pathway
VEGF functions as a dimer VEGF-A can bind to one of two receptors
The GAG heparin can influence
VEGF-A/receptor interactions
6 amino acids at the C-terminus determines whether NP-1 is engaged
and whether VEGF-A isoform is pro- or anti-angiogenic
Anti - SLTRKDPro - CDKPRR
major form expressed at basal levels
in many adult tissues
VEGF in purple and yellow
VEGF Receptor Signaling
VEGF receptor is a member of the receptor tyrosine kinase family
Ligand binding to extracellular domain induces intracellular kinase activity and subsequent downstream signaling pathways
neuropilin 1
C-terminus
C-terminus
not all of these signaling pathways occur in the same cell at the same time - context
VEGF Signaling to Endothelial Cells Promoting Angiogenesis: Migration of Tip Cells
before initiating any substantive movement toward VEGF gradient endothelial cells have to deal with
the basal laminaphenotypic change in endothelial
cells, enhanced expression of matrix degrading proteases and
enhanced migratory behavior
epithelial mesenchymal
tip cells undergo a
epithelial to mesenchymal transition to
initiate angiogenesis
Tip cells are highly motile, but non-proliferative; there is roughly one tip cell per spout
matrix-metalloproteases
Some of the Phenotypic Characteristics of Cells Involved in Angiogenesis are Shared by Metastatic
Tumor Cells
blood vessel lining
1 2
3
cells undergo a epithelial to
mesenchymal transition to
promote metastasis
broad front of migration
slender projections extending from leading edge of lamellipodia, driven
by actin polymerization
cytoskeletal rearrangements needed for directional migration regulated by small
GTPase Rho/Rac
integrins found at tips of filopodia help extensions
adhere and pull cells forward
matrix metalloproteases help tips move through barriers
integrins at trailing edge need to dissociate
Migratory Behavior of Tip Cells
gene expression needed for motility driven by
transcription factors like SRF
anchoring points to substratum, focal adhesions
Endothelial Cells of the Stalk have Phenotypes Distinct from Endothelial Tip Cells and Quiescent
Endothelial Cells
stalk cells are proliferative, but
lack sprouts
Interaction of Dll4 with notch represses VEGFR2 expression in stalk cells
suppressing the formation of new
spouts
tip cells have sprouts but much
reduced proliferation
Dll = delta-like ligand
Notch can also repress expression
colon adenocarcinoma metastasized to the lung
central areas of necrosis caused by anoxia as tumor
outgrows blood supply
Metastases may Retain Characteristics of Original Tumor and also Require a Blood Supply as Tumors Grow
Normal Angiogenesis is a Careful Balance of Pro- and Anti-Angiogenic Stimuli
This is not the case for tumor vasculature where pro-angiogenic signals predominate
creating vasculature that is anything but normal
angiostatin – inhibitor of
angiogenesis
Angiogenesis as a Target of Cancer Chemotherapy
M. Judah Folkman major proponent of using this approach to treat cancer
bevacizumab – antibody that recognizes and binds VEGF
• used alone to treat glioblastoma refractory to other treatments
• used in combination with other drugs to metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer
Strategies for Anti-Angiogenesis Therapy
sorafenib – small molecule inhibitor of VEGFR-2 – tyrosine kinase inhibitor
Suffixes in drug development
mab – antibody-based therapy
nib – small molecule kinase inhibitors
(u)
humanized antibodies
A Notorious Angiogenesis Inhibitor
thalidomide lenalidomide
currently approved for use in multiple myeloma and myelodysplastic
syndromes
tumor vessels lack normal arterial venous hierarchy, and are leaky and often
insufficient
this can impede drug delivery
anti-angiogenic therapy can
normalize tumor vasculature and improve delivery of other drugs
Effects of Normalizing Tumor Vasculature in Anti-Angiogenesis Therapy
normalized vasculature may reduce metastasis
A Bevacizumab “Success” Story
J. Clin. Onc. (2007) 25, 1539-1544
829 patients, metastatic colon cancer, treated with standard therapy or standard therapy + bevacizumab
Standard therapy is FOLFOX
• Fluorouracil• Leucovorin• Oxaliplatin
OH H
OCH2
N
N
O
O
+
OH H
OCH2
N
N
O
O
H3C
+
O – P - O
O
O-
O – P - O
O
O-
N
N
H2NN
N5
O H2C
CH2
H
N10
R
N5,N10-methylene tetrahydrofolate
N
N
H2NN
N5
OCH2
N10
R
dihydrofolate
dUMP
dTMP
thymidylate synthase
Thymidylate Synthesis
• C6 is in the β position of an αβ unsaturated ketone and so is susceptible to a nucleophilic attack
6
H
• H5 of uracil exchanges with solvent• H6 of N5N10 –THF transfers to the C5 methyl group
Thymidylate Synthase: Reaction Mechanism
N
CN
C
C
C
12
34
5
6
O
O
H
U
dRib
F
H+
Fluorine is the most electronegative atom and will not be released as F+ consequently the enzyme is trapped in a covalent ternary complex with substrate and cofactor
Fluorodeoxyuracil is used clinically as a anticancer drug. It selectively kills rapidly dividing cells making thymidine.
suicide inhibitor
Side effects include anemia, infections, nausea, and hair loss
N
CN
C
C
C
O
O
HN
CN
C
C
C
O
O
H F
HH
H
F
Hdihydropyrimidine dehydrogenase
or uracil /thymidine
Defects in Pyrimidine Catabolism can Cause Life-Threatening Toxicities when Using 5-Fluoruracil
pharmacogenomics
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%
Limitations of Anti-Angiogenic Therapy for Cancer
• Many tumors overcome effects of targeted anti-angiogenic therapies by up-regulating alternative pathways
other growth factors unrelated to VEGF can also promote angiogenesis, eg fibroblast derived growth factor and platelet derived growth factor
• Hypoxia may promote more invasive phenotypes in tumor cells
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
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
Fully Human Monoclonal Antibodies (The Adalimumab Story)
Goal: develop an antibody to treat inflammatory diseases
• ~ 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
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
Encouraging Data from First Phase I Trail for Infliximab
CRP: C reactive protein, a marker for inflammation
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
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
Approaches to Isolation of Fully Human Antibodies: 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
top related