Rituxan mediated B-cell depletion involves three complementary mechanisms: complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and a synergistic effect of serum augmenting ADCC (S-ADCC). Experimental data shows CDC and ADCC do not exclude each other and act in sequence. A mathematical model of this trifold sequential lysis process accurately fits the data. The model indicates greatest sensitivity of B-cell depletion to CD20 expression levels for more expressive lymphoma cells like those in Waldenstrom Macroglobulinemia. It also shows serum constituents are important for the total lysis outcome of Rituxan therapy.

1. About Rituxan mediated B-cell depletion in human serum:
Is there synergy between complement & effector cells?
Colin M Perrott
CDC effectiveness depends on CD20 expression and is never total
ADCC is about 50% effective without serum for all CD20 levels
CDC + ADCC in human serum is augmented by 1.63±0.36
Serum augments ADCC effectiveness of mAb targeted cells
Rituxan monotherapy:
Analysis of in vitro Lysis studies using PMBC’s
1

2. Abstract
Reported studies in vitro of Rituxan mediated B-cell depletion have suggested that
complement and effector cells (primarily NK cells) in peripheral mononuclear blood
have compensating or complementary action. The studies showed PMBC’s can
eliminate a population of cells resistant to Rituxan mediated CDC. Similarly,
complement can eliminate cells resistant to ADCC in vitro. This indicated a conjoint
action of complement and effector cells by some undefined mechanism.
We have attempted to quantify the conjoint action. We conclude that observed
synergy of CDC and ADCC rates occurs by serum augmentation of mAb induced
ADCC in the presence of serum compared to a culture media based on IL-2. Its
strength is proportional to the ADCC rate achieved by effector cells in media.
The model shows that greatest sensitivity of B-cell depletion effectiveness to CD20
expression is associated with the most expressive lymphoma cells, including those
typical of Waldenstrom Macroglobulinemia (WM) . This is a range of B-cells where
(1) total lysis efficiency is at an intermediate level,
(2) efficiencies among the component processes are comparable, and
(3) the quantifiable dynamic sensitivities are similar.
April 2009 About Rituxan in Human Serum: 2
Colin M Perrott

3. Rituxan did not eliminate all B-cells in serum
Its effectiveness depends on the CD20 expression level.
MODEL OF CDC & ADCC SUPERPOSITION
1
0.9 ADCC in media
Cell Survival Probability
CDC by serum
0.8 Lysis in Serum
0.7 Idealized Process
0.6
0.5
0.4
0.3
0.2
0.1
0
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cells express ABS CD20 in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 3
Colin M Perrott

4. Rituxan mediated ADCC and CDC are complementary in their action.
Data – van Meerten et al. (2006)
TRIALS USING HUMAN SERUM SHOWED THAT:
DIRECT APOPTOSIS BY RITUXAN IS MINIMAL.
SOME CELLS ARE RESISTANT ALTHOUGH
THEY ARE POSITIVE TO RITUXAN.
CELLS RESISTING ADCC ARE ELIMINATED
BY SEQUENTIAL CDC, AND VICE VERSA.
THEREFORE WE DEVELOPED A MODEL IN WHICH:
A TRIFOLD LYSIS MECHANISM MAY OPERATE.
ACTION IS COOPERATIVE, NOT COMPETITIVE.
CONTRIBUTIONS MAY BE EVALUATED FOR
EFFICIENCY AND SENSITIVITY TO INDENTIFIABLE
DYNAMIC VARIABLES
April 2009 About Rituxan in Human Serum: 4
Colin M Perrott

5. RITUXAN MEDIATED CELL LYSIS
Experimental observations
-------------------------------------------
CDC in serum:
Kill effectiveness depends on CD20 expression.
Experiences opponent factors e.g. CD59.
Can achieve 100% kill of normal B-cells.
-----------------------------------------
ADCC in culture media:
Kill efficiency is insensitive to CD20 expression.
ADCC in culture media achieves ~50% kill level.
Extended time does not complete lysis.
-----------------------------------------
ADCC + CDC in serum:
Shows greater potency due to some undefined
synergy mechanism.
1. Is there interdependence of ADCC and CDC?
2. Is ADCC enhanced by the serum?
3. What signaling pathway is stimulated?
Source: Van Meerten et al, Clin Cancer Res (2006) 12: 4027-4025
April 2009 About Rituxan in Human Serum: 5
Colin M Perrott

6. Rituxan mediated ADCC and CDC are complementary in their action.
Data – van Meerten et al. (2006)
Ab Initio - THIS IMPLIES A DUAL COMPONENT SEQUENTIAL PROCESS BUT THE DATA GIVE
COMPELLING EVIDENCE FOR A THIRD measurable COOPERATIVE PROCESS.
REFERENCE
Survival rate =99.9 ± 7.4% ∴ Direct Apoptosis → insignificant
Control value is Rituximab alone:
April 2009 About Rituxan in Human Serum: 6
Colin M Perrott

7. What might the cooperative mechanism involve?
Propositions:
The required binding sites for ADCC pre-exist on cells resisting CDC
The required binding sites for CDC pre-exist on cells resisting ADCC
Either;
There is a process with symmetrical signaling dependence on complement
and effector cells ( IL-2 stimulated NK cells) with respect to the mAb, or
Serum augments the mAb induced ADCC kinetics ( ‘S-ADCC’ ).
Model Features:
The synergy effect has constant magnitude for all CD20 expression
ADCC effectiveness is constant for the range of CD20 expression
The detail argues for an S-ADCC effect.
Model Outcomes:
Complement is essential to cell lysis at all CD20 expression levels
CDC is the prime mechanism at CD20 higher than ~ 650K/cell
The S-ADCC is effective at all CD20 levels exhibited by WM
The S-ADCC is probably due to NK cell cytotoxicity being heightened by a
component of the serum…most likely DHEAS
April 2009 About Rituxan in Human Serum: 7
Colin M Perrott

8. The “Synergy” of ADCC + CDC:
The cytotoxic efficiency of NK cells in serum is enhanced x 1.63 ± 0.36
compared to culture medium (where NK cells were stimulated by IL-2).
We are able to fit the experimental data very closely---
B-Cell Survival vs Rituxan
Data from van Meerten (2006)
CDC
ADCC
100.0
ADCC+CDC
Fit [ADCC*CDC]
80.0
Survival Rate (%)
60.0
40.0
20.0
0.0
400 450 500 550 600 650 700
MFI CD20 Expression
April 2009 About Rituxan in Human Serum: 8
Colin M Perrott

9. There aren’t many options for an
event model
The results show that CDC and
ADCC do not exclude each other,
acting as alternatives for cell lysis
9

10. INDEPENDENT LYSIS MECHANISMS: The survival rates add.
The total probability of cell survival is Γ = a ⋅ μ + b ⋅ λ + c ⋅ η
The total probability of cell death is Ψ = 1 − Γ since a + b + c = 1
Viable Survivor Cell
μ
1−μ Cell Lysis by CDC
a =α-β
Rituxan
1−λ
Direct Apoptosis
b =1-α
λ
Viable Survivor Cell
c=β 1−η
Cell Lysis by ADCC
η
a+b+c=1
Viable Survivor Cell
This does not fit with the available data
April 2009 About Rituxan in Human Serum: 10
Colin M Perrott

11. SEQUENTIAL LYSIS MECHANISMS: The survival rates multiply.
Γ1 = (α − β ) ⋅ μ1 ⋅ η 2 Γ2 = β ⋅ μ 2 ⋅ η1
and
∴ Γ1+ 2 ≡ Γ1 + Γ2 = α ⋅ μ1 ⋅ η 2 + β ⋅ ( μ 2 η1 − μ1 η 2 ) → αμη μ 2 η1 = μ1 η 2
if
The total probability of cell survival is Γ = (1 − ρ ) + Γ1+ 2 → α ⋅ μ ⋅ η when ρ ≈ 1
The total probability of cell death is Ψ = ρ − Ψ1+ 2 → 1 − α ⋅ μ ⋅ η
1−μ1 1−η2 ADCC
CDC
α−β
Rituxan Viable Cell
Γ1
η2
μ1
η1
μ2
β Viable Cell
Γ2
1−μ2
1−η1
CDC
ADCC
ρ−α
The data for this trial indicate: Direct Apoptosis
ρ→1 1−ρ
no direct loss
α→0.999
nil apoptosis Unaffected Cell
April 2009 About Rituxan in Human Serum: 11
Colin M Perrott

12. TRIFOLD SEQUENTIAL LYSIS MECHANISMS: CDC, ADCC, S-ADCC
Γ1+ 2 → α ⋅ μ ⋅ η ⋅ κ
→ μηκ if α ≈ 1
Total probability of cell survival is when μ 2 η 1κ 2 = μ1 η 2κ 1 = μηκ
Ψ1+ 2 → 1 − α ⋅ μ ⋅ η ⋅ κ
→ 1 − μηκ if α ≈ 1
Total probability of cell death is
ADCC
1−κ1
1−μ1 CDC
1−η2
C-ADCC
1−β
Rituxan
Viable Cell
Γ1
η2
κ1
μ1
η1 κ2
μ2
Viable Cell
β
Γ2
1−η1 1−μ2 1−κ2
CDC C-ADCC
ADCC
Fitting data to sigmoidal curve for CDC gives;
Γ1 = (1 − β ) ⋅ μ 1 ⋅ η 2 ⋅ κ 1 Γ2 = β ⋅ μ 2 ⋅ η 1 ⋅ κ 2
and
η = 0.53 ± 0.14; η κ = 0.33 ± 0.10; κ = 0.65 ± 0.18
∴ Γ1+ 2 ≡ Γ1 + Γ2 = μ 1 ⋅ η 2 ⋅ κ 1 + β ⋅ ( μ 2 η 1 ⋅ κ 2 − μ 1 η 2 ⋅ κ 1 )
April 2009 About Rituxan in Human Serum: 12
Colin M Perrott

13. Do complement and effector cells cooperate?
The killing rate for Rituxan in complete serum is elevated 1.63 ± 0.36 fold
ADCC in culture media has cytotoxic efficiency 47.2 ± 13.8%
S-ADCC in human serum has cytotoxic efficiency 67.6 ± 8.5%
Complement may not be the lone assistant in serum
There is no significant trend of enhancement with CD20 level.
B-cell Lysis in Rituxan Monotherapy
100
90
80
Percent Cell Death
70
60
50
40
30
20
10
0
400 450 500 550 600 650 700
Apoptosis CDC in serum
MFI CD20 Expression ( Thousands / cell )
ADCC in m edia S-ADCC
April 2009 About Rituxan in Human Serum: 13
Colin M Perrott

14. There is quite a lot we can
understand just from the math
The results show that CDC and
ADCC do not exclude each other,
acting as alternatives for cell lysis
14

15. Now we can look at the sensitivities
By fitting the data to a sequential process model, we gain a tool
to investigate the underlying mechanistic architecture of
influencing factors such as trends with CD20 expression
We can look at which features give the largest effects
We can look at the rate of change that might occur if the CD20
expression changes
We can see how effector cell dependent depletion processes change
the net lysis performance
We can do the same analysis for the overall effect of complement or
serum dependent processes
Finally, we can infer the sensitivity of lysis to changes in the cell
depletion efficiencies of the individual mechanisms.
April 2009 About Rituxan in Human Serum: 15
Colin M Perrott

16. Rituxan lysis of B-cells is parameter sensitive
Constituents of serum are very important to the total outcome of therapy
MODEL OF RITUXAN LYSIS
1
0.9
0.8
Cell Lysis Probability
0.7
0.6
0.5
0.4
0.3
ADCC in media
0.2
CDC by serum
Lysis in Serum
0.1
Idealized Process
0
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 16
Colin M Perrott

17. Rate of change with CD20 expression (change per million)
Maximum sensitivity is for the more expressive WM cells
SENTIVITY OF RITUXAN LYSIS TO CD20 EXPRESSION
4.0 1.00
0.90
3.5
Gradient of Lysis Probability
0.80
Net Lysis Probability
Grad CDC
3.0
0.70
Grad ADCC
Grad Serum
2.5 0.60
CDC by serum
Lysis in Serum
2.0 0.50
0.40
1.5
0.30
1.0
0.20
0.5
0.10
0.0 0.00
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 17
Colin M Perrott

18. Rate of change with process efficiency
Maximum sensitivity is for
♣ the least expressive WM cells
♣ effector cell dependent processes
SENSITVITY OF RITUXAN LYSIS TO PROCESS EFFICIENCY
1.00
1.00
0.90
0.90
Gradient of Lysis Probability
0.80
0.80
Net Lysis Probability
0.70
0.70
0.60
0.60
0.50
0.50
0.40
0.40
0.30
0.30
δΨ/δΕ 0.20
NK CELL TOTAL CYTOTOXICITY
0.20
δΨ/δΧ COMPLEMENT PROCESSES
0.10
0.10
Ψ
0.00
0.00
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 18
Colin M Perrott

19. Predicted gain from improved NK cell Cytotoxicity
A significant gain occurs from media to serum, but
♣ very big steps are still required from there !
MODEL: CDC + variable ADCC Cytotoxicity
1
0.9
Cell Lysis Probability
0.8 ADCC in media
Lysis in Serum
2x Cytotoxicity
0.7
4x Cytotoxicity
16x Cytotoxicity
0.6
0.5
0.4
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 19
Colin M Perrott

20. Predicted gain from improved NK cell Cytotoxicity
What matters is the number of cells surviving….
MODEL: CDC + variable ADCC Cytotoxicity
0.6
Cell Survival Probability
0.5
ADCC in media
0.4
Lysis in Serum
2x Cytotoxicity
0.3 4x Cytotoxicity
16x Cytotoxicity
0.2
0.1
0
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
ABS CD20 expression (millions)
WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell
April 2009 About Rituxan in Human Serum: 20
Colin M Perrott

21. Conclusions
Complement & ADCC change places in order of importance
Serum augments ADCC by a constant factor ~ 1.6x
The data is consistent with NK cell cytotoxicity stimulation
Overall lysis is greater for more CD20 expression on the B-cells
Greatest lysis sensitivity to all factors occurs at ~ 575,000 CD20 /cell
Total elimination of lymphoma B-cells will likely depend on effector cells
additional to those in peripheral blood, e.g. macrophages and neutrophils
Rituxan monotherapy:
Analysis of in vitro lysis studies using PMBC’s
21