BIOTECHNOLOGICAL APPLICATIONS 6

1. 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.

2. Allora, Is There Anything Our There that can Beat
Rituximab in a Head to Head Trial?
There are at least
three mechanisms by
which therapeutic
antibodies targeting
CD20 destroy their
target cells

3. Polymorphisms in FcγRIIIa Receptor Influence the
Therapeutic Activity of Retuximab
• FCGR3A gene encodes FcγRIIIa receptor
• Fcγ receptors are found on immune effector cells that mediate
Antibody-Dependent Cell Cytotoxicity (ADCC)
• FcγRIIIa is polymorphic at position 158 – Val or Phe
56%
35% P = 0.2
49 patients
with B cell
follicular
lymphoma
Cartron et al (2002) Blood

4. Finding Statistical Differences by Looking at Data in a
Different Way
CRu, complete
remission
unconfirmed

5. Interaction of Immunoglobulins with FcγRIII
Receptors
polymorphic position 158
Ag
Ag
bivalent

6. Cold turkey – describes the actions of a person who
abruptly gives up a habit or addiction rather than
gradually easing the process
Immunoglobulins are Subject to N-Linked
Glycosylation

7. 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

8. Cotranslational Addition of the Core Oligosaccharide
oligosaccharide transferase
signal
peptide
cytoplasm
rough ER
membrane
ER lumen
complex
high mannose
Asn-X-Ser
Asn-X-Thr

9. 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

10. Antibodies are Glycoproteins and Glycosylation can
Influence Effector Function
Antibodies are subject to N-
linked glycosylation at residue
297 of the Fc region

11. Modifying Glycosylation Patterns
Biantennary, fucosylated complex oligosaccharide
• 􏰁1,4-N-acetylglucosaminyltransferase III (GnT-III)
GluNAc
Man
Fuc
• 􏰁Man-2
Transfect cells with:
After glyco-engineering, bisected non-
fucosylated antibody

12. Kinetic Constants for the Binding of Antibodies with
Modified Carbohydrates to FcγRIIIa
20% of patients are
homozygous Valine
35% of patients are
homozygous
Phenyalanine
Biacor Traditional
equilibrium
binding assay
• GA101 with native glycosylation pattern binds much
better to polymorphic variant with Val
• GA101 with modified glycosylation pattern binds to
either polymorphic variant with roughly equal
affinity

13. 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)
Added one additional mutation
(V for L) in the hinge region
Selected on the basis of ADCC
rather than affinity

14. GA101 Does Not Cause CD20 to Aggregate in Lipid
Rafts Like Rituximab
Antibodies to CD20 fall into two classes
• Class 1 – includes Rituximab causes CD20 to aggregate in lipid rafts
• Class 2 – no aggregation, fewer molecules bound per cell
G R

15. Rituximab is more Effective than GA101 at
Promoting Complement-Dependent Cell Lysis
rituximab
GA101

16. GA101 is more Effective at ADCC and B Cell
Depletion than Rituximab
GA101
rituximab

17. GA101 is more Effective than Rituximab in Mouse
Xenograph Models of Diffuse B Cell Lymphoma
only
rituximab
Rituximab
or GA101

18. GA101 is More Effective than Rituximab in Depleting B
Cells in Clonus Monkeys
G R

19. Of Trumps Rituximab in a Clinical Trial for Chronic
lymphocytic Leukemia

20. One Last Look at Where We would be Without
Therapeutic Antibodies

21. Imatinib and Beyond: Protein Kinase
Inhibitors as Cancer Therapeutics

22. Protein Kinases as Drug Targets
Problem:
There are over 500 protein kinases encoded by the
human genome. Can a drug target just one?
Cancer Drug Gleevec (Imatinib) Wins Lasker Award
Posted at 3:43 pm CT on September 14, 2009
The shortened "Philadelphia chromosome" seen in certain leukemias (picture from
nature.com)
The big science news of the day was the announcement of the Lasker Awards, informally
thought of as the American version of the Nobel Prize for physiology and medicine. This
year’s clinical medical research award went to a trio of researchers from Oregon Health &
Science University, Sloan-Kettering Cancer Center and drug company Novartis, but you
could just as easily say it was awarded to a drug: the cancer treatment Gleevec (imatinib)
No/yes
sufficient specificity
for clinical benefit

23. Imatinib and Related Drugs Target Philadelphia
Chromosome Positive CML
• BCR-ABL fusion creates a dys-regulated
protein kinase that drives the proliferation
of CML clones
protein kinase active site
bind ATP
bind target protein
substrate
imatinib
specific kinase inhibition
A priori, targeting the protein
substrate binding site might be
expected to provide more
specificity, but…………………

24. The Imatinib Experience
• 82% of patients achieve a complete cytogenetic response (65-85%)
• majority achieve a major molecular response (40-60%)
• 7 year overall survival rate 90%
• event free survival rate 81% (65-85%)
Positives
results from the International Randomized Study of Interferon
Versus STI571 (IRIS) trial 7-year follow up
Blood (2008) 112, 186
various studies from 2003-2008
• Imatinib, however, does not destroy the cancer stem cell clone
• If you remove imatinib, CML returns
• Imatinib is the perfect drug because once you are on it, you are on
it forever
If you place a cancer cell under selective pressure for long enough without killing
it, what will happen?