BIOTECHNOLOGICAL APPLICATIONS 2

2. CD20 is an Excellent Target for a Therapeutic
Antibody to Treat B Cell Hyperproliferative Disorders
CD = Clusters of
Differentiation
(Classification
Determinant)
antigens
Early progenitors do NOT
express CD20, so B cell-
mediated adaptive immunity
could be restored after therapy

3. Mice immunized weekly with a human lymphoblastoid cell line (SB)
Spleens harvested from mice expressing high titers of anti-CD20 (polyclonal)
Hybridomas formed from spleen lymphocytes and a mouse myeloma cell line SP2/0
125I-antiCD20(murine) + hybridoma culture supernatant + SB cells
recover SB cells, wash,
monitor radioactivity
hybridomas secreting anti-
CD20 should inhibit 125I-
binding
Screening Hybridomas for Specific Monoclonal
Antibodies (circa 1994)
Reff et al (1994) Blood 83:435-445
competition assay
patent, commercializationPrevious antibody

4. Modern Approaches to Antibody Screening
ELISA Enzyme-Linked ImmunoSorbant Assay
luminol
horseradish peroxidase
made by hybridomas
goat anti-
mouse

5. Mice immunized weekly with a human lymphoblastoid cell line (SB)
Spleens harvested from mice expressing high titers of anti-CD20
Hybridomas formed from spleen lymphocytes and a mouse myeloma cell line SP2/0
125I-antiCD20(murine) + hybridoma culture supernatant + SB cells
recover SB cells, wash,
monitor radioactivity
hybridomas secreting anti-
CD20 should inhibit 125I-
binding
Screening Hybridomas for Specific Monoclonal
Antibodies (circa 1994)
Reff et al (1994) Blood 83:435-445

6. Eukaryotic Expression Vector used for the Expression
of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
antibiotic selection in E. coli
ampicillin

7. The AMP Gene Encoding β-Lactamase is used as a
Selectable Marker in Bacterial Cells
β-lactamase
cleaves this bond
ampicillin

8. Eukaryotic Expression Vector used for the Expression
of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
neomycin resistance,
selectable marker in
mammalian cells

9. Eukaryotic Expression Vector used for the Expression
of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
SV40 origin
of replication

10. Eukaryotic Expression Vector used for the Expression
of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
dihydrofolate reductase
grown in the present of
methotrexate, will
increase gene dosage
DHFR
Low [ ] methotrexate
DHFR2-3
DHFRX
Higher [ ]

11. Eukaryotic Expression Vector used for the Expression
of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20 light chain

12. CMV
promoter
consensus signal
peptide for
human
immunoglobulins
mouse
light chain
variable
region
human
light chain
constant
region
PCR amplified from
selected hybridoma
Creation of Mouse/Human Chimeric Monoclonal
Antibodies (Light Chains)
BGH
polyadenylation
signal
similar construct used for heavy chain

13. Antibody Purification
Protein A from Staphylococcus aureus binds up to 5
molecules of IgG through the Fc domain. This is
the opposite orientation an IgG would normally
bind to a pathogen surface.
Prot A Prot A Prot A
serum
IgG
flow through-
other stuff
purified IgG
washload
elute – low pH
elution buffer
(pH2.8)
A
The interaction of Protein A with IgG in this
manner disrupts opsinization

14. Antibody Characteristics: Scatchard Plot of
Equilibrium Binding Data
B/Lf = -B/Kd + RT/Kd
Plot B/Lf versus B
RT/Kd
C2B8
2B8 (murine)

15. Antibody Characteristics: Competitive Binding Assay
Ability of different monoclonoal antibodies to inhibit the
binding of 125I-CD20 (murine) to SB lymphoblastoid cells
C2B8
2B8 (murine)

16. Antibody Function: Directing Complement Binding to
Antigens
Antibody +
lymphoblastoid cells +
fluorescently-labeled
human complement
FACS (fluorescence-
activated cell sorting)
analysis
murine monoclonal Ab
humanized monoclonal Ab

17. Antibody Function: Orchestrating Cell Lysis
load SB cells with 51Cr
mix with antibody and
a source of human
complement
monitor 51Cr release
murine monoclonal Ab
humanized monoclonal Ab
load SB cells with 51Cr
mix with antibody and
effector cells (peripheral
mononuclear cells)
monitor 51Cr release
humanized monoclonal Ab
CDCC – complement dependent cell lysis ADCC – antibody dependent cell cytotoxicity

18. Antibody Function: Depleting B Lymphocytes in a
Non-Human Primate Model
• Infuse Clonus monkeys
with humanized Ab
• Injections weekly for 4
weeks
• Assess levels of B
lymphocytes 36 days
after last injection

19. Antibody Function: Selectivity
• Infuse Clonus monkeys
with humanized Ab
• Injections weekly for 4
weeks
• Assess levels of T
lymphocytes 36 days
after last injection

20. B Cell Lymphoma
US Statistics 2012
• 70,130 new cases
• 18,940 deaths
Diffuse large cell B Cell Lymphoma most prevalent of Non-
Hodgkins Lymphoma (~30%) and is considered aggressive
and treatment necessary

21. Putting the R in R-CHOP: Rituximab Clinical Trials
A four drug chemotherapeutic cocktail given the acronym CHOP was the
standard of care for non-Hodgkins B cell lymphoma until the early-2000’s when
rituximab was added. R-CHOP has since been the standard of care
C - Cyclophosphamide
H – Hydroxy-daunomycin
O – Oncovin (vincristine sulfate)
P – Prednisone
Vincristine’s most common side effects are:
peripheral neuropathy, suppression of bone
marrow activity, constipation, nervous system
toxicity, nausea, and vomiting.

22. N
Cl CH2CH2
Cl CH2CH2
P O
N
O
CH2
CH2
CH2
N
Cl CH2CH2
Cl CH2CH2
P
O
NH2
OH
phosphoramide
mustard
CH2 C C O
H
H
acrolein
hepatic p450
highly reactive causes cystitis –
treat with 2-mercapto-ethane-
sulphonate (MESNA)
N
CH2CH2
CH2CH2
P
O
NH2
OH
G
G
Nitrogen Mustards
Cyclophosphamide (Cytoxan)
can be given orally
bioactivation
pro-drug

23. Doxorubicin - anthracycline
Daunorubicin has antimitotic and cytotoxic activity through a number of proposed
mechanisms of action. Daunorubicin forms complexes with DNA by intercalation between
base pairs.
• Inhibits the ability of topoisomerases to religate cleaved DNA, causing single and
double-stranded breaks in DNA
• interferes with replication
• generates reactive oxygen species
Mechanism of action
• Daunorubicin may cause serious or life-threatening heart problems at any time during
your treatment or months to years after your treatment has ended.
Side Effects
oncologic cardiology
Anthracyclines

24. CHOP Chemotherapy Plus Rituximab Compared with CHOP
Alone in Elderly Patients with Diffuse Large-B-Cell Lymphoma
N. Engl. J. Med. (2002) 346:235
What does it mean that the patients were
randomized?
Performance status: all patients listed as
good to fair
Prognostic index: higher score, higher
risk of death: combination of disease
stage, performance status and LDH
levels
B symptoms: weight loss, fever, night
sweats

25. Trial Outcomes (24 Month Follow Up)
Rituximab reduced the
relative risk of an event by
42%

26. Event Free Survival: CHOP Versus R-CHOP
median time to event not reached
in R-CHOP arm
median time to event 13 months in
CHOP arm

27. Overall Survival: CHOP Versus R-CHOP
70%
57%

28. Adverse Effects: CHOP Versus R-CHOP
Infusion-related
grade 1 events
most adverse events
associated with known
CHOP toxicities

29. In conclusion, the addition of rituximab to CHOP chemotherapy,
given for eight cycles to elderly patients with newly diagnosed
diffuse large-B-cell lymphoma, significantly increases the rate of
complete response, decreases the rates of treatment failure and
relapse, and improves event-free and overall survival as compared
with standard CHOP alone. These gains were achieved without a
significant increase in clinically significant toxic effects.

30. Trial Outcomes (10 Year Follow Up)
Blood (2010) 116:2040
Most related to underlying conditions
prior to lymphoma diagnosis

31. median time to progression 4.8
years in R-CHOP arm
median time to progression 1.2
years in CHOP arm
Progression Free Survival Long Term: CHOP Versus
R-CHOP

32. Disease Free Survival in Patients Who had
Originally had a Complete Remission
median time to event not reached
in R-CHOP arm
median time to event 3.4 years in
CHOP arm
These findings confirm that the use of R-CHOP can improve patient outcomes in elderly
DLBCL patients, and that the beneficial effects are sustained over a long follow-up period.

33. Rituximab’s Success has Opened the Floodgates of
Further Developments in Therapeutic Antibody
Development
Refinements on Rituximab
Antibodies against other targets for use in different cancers
FDA approved therapeutic antibodies circa 2006

34. Rituximab’s Success has Opened the Floodgates of
Further Developments in Therapeutic Antibody
Development
Continued refinement
Rituximab
CDR – complementarity
determining region

35. Light chain
0 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19
20 21 22 23 24 25 26 27
27A 27B 27C 27D 27E 27F 28 29
30 31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49
50 51 52 53 54 55 56 57 58 59
60 61 62 63 64 65 66 67 68 69
70 71 72 73 74 75 76 77 78 79
80 81 82 83 84 85 86 87 88 89
90 91 92 93 94 95
95A 95B 95C 95D 95E 95F 96 97 98 99
100 101 102 103 104 105 106
106A 107 108 109
L1: L24-L34
L2: L50-L56
L3: L89-L97
H1: H26-H35B
H2: H50-H65
H3: H95-H102
Sequence gazing:
• starts approximately at residue 24
• residue before always a cysteine
• residue after always a tryptophan
Length 10 to 17 residues
Kabat System for Identifying Complementary
Determining Regions of Immunoglobulins

36. Making a Humanized Antibody from Mouse
Complementary Determining Regions
Replace human with
mouse CDRs

37. Polymerase Chain Reaction
• Starting DNA is rapidly diluted out with each cycle of
PCR
• After several cycles, newly synthesized DNA bounded by
forward and reverse primers is the dominant DNA
species
✓
✓

38. Using PCR for Deletion Mutagenesis
• Starting material: plasmid DNA
containing a gene of interest
• Two primers forming the boundaries of
the region that will be deleted –
directing DNA synthesis away from the
region that will be deleted
• PCR amplification of DNA excluding
the region that will be deleted
• Ligation of PCR product, confirmation
by DNA sequence analysis

39. PCR can also be Used for Substitution Mutagenesis
• Starting material: plasmid DNA
containing a gene of interest
• Two primers abutting one another facing in
opposite directions
• One or both primers have desired substitutions but
retain sufficient complementarity to anneal to
human sequences
• PCR amplification of DNA: DNA with
altered sequence will eventually
predominate
• Ligation of PCR product, confirmation
by DNA sequence analysis

40. Making a Humanized Antibody from Mouse
Complementary Determining Regions
Replace human with
mouse CDRs

41. Three-Dimensional Structure of the Variable Regions
of Heavy and Light Chains

42. Determining Dissociation Constants Using Surface
Plasmon Resonance (Biacore Biosensors)
• gold layer has a characteristic angle of reflected light
• angle altered by binding interactions on the surface
exposed to flow cell
flow cell
immobilized

43. Biacore Readout
On rate
Off rate

44. Three-Dimensional Structure of the Variable Regions
of Heavy and Light Chains

45. Refining the Properties of Humanized Antibodies

46. Pharmacokinetics of Humanized Antibody
Against VEGF
• Half life in humans 17-21 days
• No evidence of anti-drug antibodies

47. Humanized Antibody has Anti-Tumor Properties in a
Pre-Clinical Model
Antibody raised to VEGF
VEGF: vascular
endothelial growth factor

48. Targeting Angiogenesis in Cancer Chemo- and
Immuno-Therapy
Folkman (1971, NEJM 285,1182) rather than target tumor
cells themselves attack the tumor’s supply of nutrients and
other factors needed for survival
• New capillary growth (for tumors) is even more vigorous
and continuous than a similar outgrowth of capillary
sprouts observed in fresh wounds or inflammation
• The growth of solid neoplasms is always accompanied
by neovascularization
• Targeting normal cells supporting tumor cell growth may
be more efficacious than targeting genetically unstable
tumor cells
• Target for anti-angiogenesis therapy - VEGF