Rituxan mediates B-cell depletion through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Rituxan is not fully effective on its own and human serum enhances its potency. A model was developed showing that CDC, ADCC, and a serum augmentation of ADCC (SAM) act cooperatively rather than competitively. The synergy between these mechanisms was quantified, with human serum augmenting ADCC by 1.63-fold on average. Sensitivity analysis revealed that lysis is most sensitive to changes in CD20 expression levels and ADCC efficiency for B-cells expressing intermediate CD20 levels typical of Waldenstrom Macroglobulinemia.

1. About Rituxan mediated B-cell depletion in human serum:
What is the 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 mAb effectiveness by ADCC
Rituxan monotherapy:
Analysis of in vitro Lysis studies using PBMC’s
1

2. Abstract
Reported studies in vitro of Rituxan mediated B-cell depletion demonstrate that
Complement and peripheral mononuclear blood (PMBC) characterized by strong NK
cell activity have compensating action against cells found to be resistant to one or
other process. The 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 (SAM), rather than complement specifically. Its
strength is proportional to the underlying ADCC rate achieved by effector cells alone.
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.
March 2009 About Rituxan in 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 Pure ADCC
Cell Survival Probability
Pure CDC
0.8 PMBC's 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
March 2009 About Rituxan in 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:
THERE IS A TRIFOLD LYSIS MECHANISM.
ACTION IS COOPERATIVE, NOT COMPETITIVE.
CONTRIBUTIONS MAY BE EVALUATED FOR
EFFICIENCY AND SENSITIVITY TO INDENTIFIABLE
DYNAMIC VARIABLES
March 2009 About Rituxan in Serum 4
Colin M Perrott

5. RITUXAN MEDIATED CELL LYSIS
B-cells in human serum are not
depleted fully by Rituxan
-------------------------------------------
CDC in serum depends on CD20 expression.
CDC experiences opponent factors e.g. CD59.
CDC achieves 100% kill of normal B-cells.
-----------------------------------------
ADCC excluding serum is insensitive to CD20 level.
ADCC achieves about 50% kill efficiency.
Extended time does not complete lysis.
-----------------------------------------
ADCC + CDC in serum shows greater potency.
1. Is this a synergy effect between ADCC and CDC?
2. Is ADCC enhanced by the serum?
Source: Van Meerten et al, Clin Cancer Res (2006) 12: 4027-4025
March 2009 About Rituxan in 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 COOPERATIVE PROCESS.
REFERENCE
Survival rate =99.9 ± 7.4% ∴ Direct Apoptosis → insignificant
Control value is Rituximab alone:
March 2009 About Rituxan in 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 with respect to the mAb, or
Serum augments the mAb induced ADCC processes (SAM).
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 a SAM effect- possibly involving passive immune
complexes and/or binding of mAb to FcγRIIb or complement to CR2 on B cells.
Model Outcomes:
Complement is essential to cell lysis at all CD20 expression levels
CDC is the prime mechanism at CD20 higher than ~ 650K/cell
SAM is effective at all CD20 levels exhibited by WM
March 2009 About Rituxan in Serum 7
Colin M Perrott

8. Do complement and effector cells cooperate?
The killing rate for Rituxan in complete serum is elevated 1.63 ± 0.36 fold
Complement may not be the lone assistant in serum
Serum augmented ADCC average efficiency is 67.6 ± 8.5%
ADCC without serum has efficiency 47.2 ± 13.8%
There is no significant trend of SAM with CD20 expression 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
MFI CD20 Expression ( Thousands / cell )
ADCC S-ADCC
March 2009 About Rituxan in Serum 8
Colin M Perrott

9. Now we can look at the sensitivities
By fitting the data to a sequential process model, we can investigate
the trends with CD20 expression and the underlying architecture
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.
March 2009 About Rituxan in Serum 9
Colin M Perrott

10. 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
Pure ADCC
0.2 Pure CDC
PMBC's in Serum
0.1
All S-Effects
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
March 2009 About Rituxan in Serum 10
Colin M Perrott

11. 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
3.0
0.70
Grad CDC
Grad ADCC
2.5 0.60
Grad Serum
Pure CDC
2.0 0.50
Human Serum
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
March 2009 About Rituxan in Serum 11
Colin M Perrott

12. 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
EFFECTOR CELL PROCESSES
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
March 2009 About Rituxan in Serum 12
Colin M Perrott

13. Rate of change with mechanism efficiency
Maximum sensitivity is for
♣ the least expressive WM cells
♣ change in the ADCC mechanism
SENSITIVITY OF RITUXAN LYSIS TO MECHANISM EFFICIENCY
1.00 1.00
Gradient of Lysis Probability
0.90 0.90
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
δΓ/δμ CDC
0.20 0.20
δΓ/δη ADCC
δΓ/δκ SAM
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
March 2009 About Rituxan in Serum 13
Colin M Perrott

14. Conclusion
Complement drives a major component of cell deletion
Data suggested that serum augments ADCC by a constant amount
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
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