About Rituxan In Human Serum

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About Rituxan mediated B-cell depletion in human serum: Is there synergy between complement & effector cells?

CDC effectiveness depends on CD20 expression and is never totalADCC is about 50% effective without serum for all CD20 levelsCDC + 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

Colin M Perrott

April 2009 Colin M Perrott About Rituxan in Human Serum: 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.


Rituxan did not eliminate all B-cells in serumIts effectiveness depends on the CD20 expression level.

WM B-cells express ABS CD20 in the range ~ 0.3 to 0.6 million/cell

MODEL OF CDC & ADCC SUPERPOSITION

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ABS CD20 expression (millions)

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ADCC in mediaCDC by serumLysis in SerumIdealized Process


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 ALTHOUGHTHEY ARE POSITIVE TO RITUXAN.

CELLS RESISTING ADCC ARE ELIMINATEDBY 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


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


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

Control value is Rituximab alone: Survival rate =99.9 ± 7.4% ∴ Direct Apoptosis → insignificant


What might the cooperative mechanism involve?

Propositions:The required binding sites for ADCC pre-exist on cells resisting CDCThe 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, orSerum augments the mAb induced ADCC kinetics ( ‘S-ADCC’ ).

Model Features:The synergy effect has constant magnitude for all CD20 expressionADCC effectiveness is constant for the range of CD20 expressionThe detail argues for an S-ADCC effect.

Model Outcomes:Complement is essential to cell lysis at all CD20 expression levelsCDC is the prime mechanism at CD20 higher than ~ 650K/cellThe 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


The “Synergy” of ADCC + CDC: The cytotoxic efficiency of NK cells in serum is enhanced x 1.63The cytotoxic efficiency of NK cells in serum is enhanced x 1.63 ±± 0.360.36compared to culture medium (where NK cells were stimulated by ILcompared to culture medium (where NK cells were stimulated by IL--2).2).

We are able to fit the experimental data very closely---

B-Cell Survival vs RituxanData from van Meerten (2006)

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MFI CD20 Expression

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CDCADCCADCC+CDCFit [ADCC*CDC]

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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


INDEPENDENT LYSIS MECHANISMS: The survival rates add.

The total probability of cell survival is The total probability of cell death is

This does not fit with the available data

μ

c = β

1−μ

η

1−η

1−λ

λ

b =1-α Direct Apoptosis

Cell Lysis by ADCCCell Lysis by ADCC

Cell Lysis by CDCCell Lysis by CDC

Viable Survivor Cell

RituxanRituxan



a =α-β

a + b + c = 1

ηλμ ⋅+⋅+⋅=Γ cba11 =++Γ−=Ψ cbasince


SEQUENTIAL LYSIS MECHANISMS: The survival rates multiply.

The total probability of cell survival is The total probability of cell death is

Unaffected Cell

CDCCDC

Direct Apoptosis

α−βRituxanRituxan

ADCCADCC

μμ11

β

11−−μμ11

η1

1−η1

μμ22

11−−μμ22

η2

1−η2

ρ−α

Viable CellΓ1

ADCCADCC

Viable CellΓ2

CDCCDC

The data for this trial indicate: no direct loss ρ→1 nil apoptosis α→0.999

1−ρ

21122112212121

122211

)()(

ημημαμηημημβημαημβημβα

=→−⋅+⋅⋅=Γ+Γ≡Γ∴

⋅⋅=Γ⋅⋅−=Γ

+ ifand

1)1( 21 ≈⋅⋅→Γ+−=Γ + ρημαρ whenημαρ ⋅⋅−→Ψ−=Ψ + 121


TRIFOLD SEQUENTIAL LYSIS MECHANISMS: CDC, ADCC, S-ADCC

Total probability of cell survival is

Total probability of cell death is

RituxanRituxan

CDCCDC

1−β

ADCCADCC

μμ11

β

11−−μμ11

η1

1−η1

Viable CellΓ1

ADCCADCC

Viable CellΓ2

CC--ADCCADCC

κκ22

11−−κκ22

η2

1−η2CC--ADCCADCC11−−κκ11

κκ11

CDCCDC11−−μμ22

μμ22

)()1(

1212121212121

21221211

κημκημβκημκημβκημβ

⋅−⋅⋅+⋅⋅=Γ+Γ≡Γ∴

⋅⋅⋅=Γ⋅⋅⋅−=Γ

+

and

121

≈→

⋅⋅⋅→Γ +

αμηκκημαif

11121

≈−→

⋅⋅⋅−→Ψ +

αμηκκημαif

μηκκημκημ == 121212when

Fitting data to sigmoidal curve for CDC gives;η = 0.53 ± 0.14; η κ = 0.33 ± 0.10; κ = 0.65 ± 0.18


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 serumThere is no significant trend of enhancement with CD20 level.

B-cell Lysis in Rituxan Monotherapy

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MFI CD20 Expression ( Thousands / cell )

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Apoptosis CDC in serumADCC in media S-ADCC

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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


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 effectsWe 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.


Rituxan lysis of B-cells is parameter sensitiveConstituents of serum are very important to the total outcome of therapy

WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell

MODEL OF RITUXAN LYSIS

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ADCC in mediaCDC by serumLysis in SerumIdealized Process


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

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yGrad CDCGrad ADCCGrad SerumCDC by serumLysis in Serum


Rate of change with process efficiencyMaximum sensitivity is for

♣ the least expressive WM cells♣ effector cell dependent processes


SENSITVITY OF RITUXAN LYSIS TO PROCESS EFFICIENCY

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δΨ/δΕδΨ/δΧ

Ψ

NK CELL TOTAL CYTOTOXICITY

COMPLEMENT PROCESSES


Predicted gain from improved NK cell CytotoxicityA significant gain occurs from media to serum, but

♣ very big steps are still required from there !


MODEL: CDC + variable ADCC Cytotoxicity

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ADCC in mediaLysis in Serum2x Cytotoxicity4x Cytotoxicity16x Cytotoxicity


Predicted gain from improved NK cell CytotoxicityWhat matters is the number of cells surviving….


MODEL: CDC + variable ADCC Cytotoxicity

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ADCC in mediaLysis in Serum2x Cytotoxicity4x Cytotoxicity16x Cytotoxicity

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ConclusionsComplement & 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

Rituxan monotherapy: Analysis of in vitro lysis studies using PMBC’s

Total elimination of lymphoma B-cells will likely depend on effector cells additional to those in peripheral blood, e.g. macrophages and neutrophils

Technology

About Rituxan In Human Serum