-
Dr. Gopichand Pendurti M.B.B.S
ACTIVITY OF RITUXIMAB AND OFATUMUMAB AGAINST MANTLE CELL
LYMPHOMA(MCL) IN VITRO IN MCL CELL LINES BY COMPLEMENT
DEPENDENT CYTOTOXICITY (CDC)AND ANTIBODY-DEPENDENT CELL MEDIATED
CYTOTOXICITY ASSAYS(ADCC)
Mentor: Dr. Francisco J. Hernandez-Ilizaliturri MD
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Overview of presentation
•Introduction to mantle cell lymphoma.
•Concept of minimal residual disease.
•Anti CD 20 antibodies.
•51Cr release assays.
•Results.
•Future.
•Flow cytometry on cell lines.
-
•Mantle cell lymphoma is characterized by abnormal proliferation
of mature B lymphocytes derived from naïve B cells.
•Constitutes about 5% of all patients with Non Hodgkin's
lymphoma.
•Predominantly in males with M:F ratio 2.7:1 with onset at
advanced age (median age 60yrs).
•It is an aggressive lymphoma with median survival of patients
being 3-4 years.
•Often presents as stage III-IV with lymphadenopathy,
hepatosplenomegaly, gastrointestinal involvement, peripheral blood
involvement.
MANTLE CELL LYMPHOMA
Pedro Jares, Dolors Colomer and Elias Campo Genetic and
molecular pathogenesis of mantle cell lymphoma: perspectives for
new targeted therapeutics Nature revision of cancer 2007
October:7(10):750-62
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•Genetic hallmark is t(11:14)(q13:q32) translocation leading to
over expression of cyclin D1 which has one of the important
pathogenetic role in deregulating the cell cycle.
•Other pathogentic mechanisms include molecular and chromosomal
alterations that Target proteins that regulate the cell cycle and
senecense (BMI1,INK4a,ARF,CDK4 AND RB1). Interfere with cellular
response to DNA damage(ATM,CHK2 and p53).
Pedro Jares, Dolors Colomer and Elias Campo Genetic and
molecular pathogenesis of mantle cell lymphoma: perspectives for
new targeted therapeutics Nature revision of cancer 2007
October:7(10):750-62
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MORPHOLOGY
•Spectrum of variants from classic type to blastoid and
pleomorphic types
Classic MCL Blastoid MCL Pleomorphic MCL
Small–medium-sized lymphocytes with irregular nuclei and
inconspicuous nucleoli
Rounded nuclei, finely dispersed chromatin and inconspicuous
nucleoli
Larger cells with irregular and pleomorphic nuclei and distinct
small nuclei
Pedro Jares, Dolors Colomer and Elias Campo Genetic and
molecular pathogenesis of mantle cell lymphoma: perspectives for
new targeted therapeutics Nature revision of cancer 2007
October:7(10):750-62
-
Immunophenotype
•Mature B-cell phenotype with moderate to strong expression of
surface immunoglobulin's (Ig M and Ig D) predominantly lambda.
•B-cell-associated antigens such as CD20, CD22, CD79, and the
T-cell-associated antigen CD5.
Pedro Jares, Dolors Colomer and Elias Campo Genetic and
molecular pathogenesis of mantle cell lymphoma: perspectives for
new targeted therapeutics Nature revision of cancer 2007
October:7(10):750-62
-
•Molecular remission is an independent prognostic factor for
response duration.
•In spite of upfront high dose chemotherapy induction with auto
stem cell transplantation about 44% of patient still have minimal
residual disease.
Pott et al. Molecular remission is an independent predictor of
clinical outcome in patients with mantle cell lymphoma after
combined immunotherapy European MCL intergroup study. Blood
.2010;115(16):3215-3223
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Pott et al. Molecular remission is an independent predictor of
clinical outcome in patients with mantle cell lymphoma after
combined immunotherapy European MCL intergroup study. Blood
.2010;115(16):3215-3223
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PB-Peripheral blood.
BM-Bone marrow.
Minimal residual disease quantification by RQ-PCR of 190
patients before, during and after induction.
Pott et al. Molecular remission is an independent predictor of
clinical outcome in patients with mantle cell lymphoma after
combined immunotherapy European MCL intergroup study. Blood
.2010;115(16):3215-3223
-
Response duration (RD) according to MRD status after combined
immunochemotherapy.
RD according to MRD status in PB and/or BM after end of
induction in MCL Younger and MCL Elderly patients
RD duration according to MRD status assessed in the PB after
induction treatment in both trials.
RD duration according to MRD status assessed in the BM after
induction treatment in both trials.
RD-Response duration MRD-Minimal residual disease
Pott et al. Molecular remission is an independent predictor of
clinical outcome in patients with mantle cell lymphoma after
combined immunotherapy European MCL intergroup study. Blood
.2010;115(16):3215-3223
-
RD according to MRD status assessed in PB and/or BM within the
first 12 months after ASCT in MCL Younger patients.
RD according to MRD status assessed in PB and/or BM during
thefirst year of maintenance in MCL Elderly patients
RD-Response duration MRD-Minimal residual disease ASCT-
Autologous stem cell transplantation
Pott et al. Molecular remission is an independent predictor of
clinical outcome in patients with mantle cell lymphoma after
combined immunotherapy European MCL intergroup study. Blood
.2010;115(16):3215-3223
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“Can the use of new anti CD20 monoclonal antibodies like
ofatumumab lead to molecular remission in patients with mantle cell
lymphoma, ultimately increasing the response duration along with
upfront high dose induction therapy and auto stem cell
transplantation?”.
RESEARCH QUESTION
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Edwards et al. Nature Reviews Immunology 6, 394–403 (May 2006) |
doi:10.1038/nri1838
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Mechanisms of Action of Anti-CD20 Antibodies
Maloney DG. N Engl J Med 2012;366:2008-2016.
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OFATUMUMAB
•TYPE I human IgG1K antibody with molecular weight of 149
Kda.
•Ofatumumab binds to novel epitope of CD20 which encompasses
small extracellular loop.
•Ofatumumab lyses Raji cells, Daudi cells better than rituximab
through CDC where as ADCC results were comparable.
•CDC with ofatumumab is not dependent on cell surface expression
of complement region molecules.
•CDC occur even at lower density of CD20 on cell surface than
with rituximab.
Bruce D. Cheson, Ofatumumab, a Novel Anti-CD20 Monoclonal
Antibody for the Treatment of B-Cell Malignancies. Journal of
clinical oncology,28(21),3525-3530.
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The binding of ARZERRA to CD20. (2011). Retrieved May 9th from
http://hcp.gsk.com/therapy_areas/oncology/arzerra/mechanism-of-action/#R4
Arzerra:Ofatumumab
Binding site of Ofatumumab to CD 20 on the B-cells
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ARZERRA induces cell lysis by CDC. (2011). Retrieved on May 9th
from
http://hcp.gsk.com/therapy_areas/oncology/arzerra/mechanism-of-action/#R4
Ofatumumab induces cell lysis by CDC
Arzerra:Ofatumumab
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• Beum et al and Taylor et al compared the c3b deposition and
cell killing by ofatumumab and rituximab.
Complement activation
Induces membrane blebbing
Generates streamers (long, thin structures)
Their extent of formation correlates with CDC
•Ofatumumab causes more rapid and greater blebbing and streamer
formation than rituximab.
•Ofatumumab is most promising in patients with CLL who have
fludarabine-and alemtuzumab-refractory disease and in those with
bulky disease who experienced treatment failure with fludarabine
therapy.
Bruce D. Cheson, Ofatumumab, a Novel Anti-CD20 Monoclonal
Antibody for the Treatment of B-Cell Malignancies. Journal of
clinical oncology,28(21),3525-3530.
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51 Cr release assays to compare the biological activity of
various monoclonal antibodies targeting CD 20 in MCL cell lines
Material and methods:
•Mantle cell lymphoma cell lines-JeKo,Mino,Rec-1,Z-138 were
used
•Radioactive 51 Cr
•Ofatumumab (10ug/ml)
•Rituximab (10ug/ml)
•Herceptin or trastuzumab (10ug/ml)-Isotype
•Serum or peripheral blood mononuclear cells.
Figure1:Extraction of PBMC from whole blood.
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•MCL cell lines were counted and centrifuged at 2000rpm for 5
minutes.
•Removed the supernatant and added 100µl of 51 Cr to the cell
pellets, incubated them for 2 hrs at 37oc,5% co2 .
•Washed the cell lines to remove excess of chromium using RPMI
media.
•Re-suspended the cells in media to get a final concentration of
106 cells/ml.
•Placed 100µl of cell suspension in each well.
•Treated cells with 50µl of oftamumab, rituximab, isotype, serum
,PBMC or media.
•Incubated for 6 hrs ,after that detergent was added to maximum
release row.
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cells +media
cells +OFA+ Serum/PBMC
cells +RIT+ Serum/PBMC
cells +media+ detergent
cells +HER2+Serum/PBMC
cells +serum/PBMC +Media
Media Ofatumumab
Herceptin Rituximab
Serum/PBMC Detergent
Model of the 96 well plate prepared for the experiments
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•Centrifuged the plate for 5 min at 2000rpm.
•Collected 100µl of supernatant ,avoided touching the bottom of
the well.
•Read the amount of radioactivity using a beta counter
reader.
•Calculated the percent lysis using the formula
[51Cr release from sample-51Cr release from control]
[51Cr release from maximum release-51Cr release from
control]
*100 % lysis ═
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FLOW CYTOMETRY
• Flow cytometry was performed on the cell lines for the
expression of CD 20 and complement inhibitory proteins (CIP)- CD 55
and CD 59.
Why Complement inhibitory proteins?
• Rituximab resistant Raji cells had increased expression of CD
55 and CD 59.
• We compared the flow cytometry results with the flow data
available on the rituximab sensitive and rituximab resistant Raji
cells.
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STATISTICS
• SPSS 16 was used for the independent t-Test to calculate the
significance of the lysis between the two anti CD 20
antibodies.
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MCL Cell line Ofatumumab Rituximab
REC-1 25.4% 4.7%
Z-138 56.4% 0.65%
Mino 65.9% 0.5%
JeKo 43.9% 13.3%
•Ofatumumab induced significantly higher levels of cell lysis
compared to rituximab in CDC assays.
RESULTS
p-value significant at
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CDC on MCL cell lines
Z-138
JeKo
REC-1
MINO
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REC-1
Z138
JEKO
MINO
CDC on MCL cell lines
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MCL Cell line Ofatumumab Rituximab
REC-1 12% 14%
Z-138 14% 12%
Mino 1% 3%
JeKo 12% 12%
•Ofatumumab and rituximab have comparable levels of cell lysis
in ADCC assays.
p value not significant - 0.264
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•Ofatumumab and rituximab showed comparable level of cell lysis
in ADCC assays
Z-138 JeKo
REC-1 MINO
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ADCC on MCL cell lines
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UnstainedFITCCD20
FL1-H
Coun
t
100 101 102 103 1040
25
50
75
100
FL1-H
Cou
nt
100 101 102 103 1040
25
50
75
100
FL1 H
Cou
nt
100 101 102 103 1040
25
50
75
100
FL1 H
Coun
t
100 101 102 103 1040
25
50
75
100
JeKo REC-1
Z138 MINO
Surface expression of CD 20 in MCL cell lines
Histograms of the flow cytometry for CD20 surface
expression.
-
Raji.031
FL1-H
Cou
nt
100 101 102 103 1040
25
50
75
100
Raji4RHP10-2.034
FL1-H
Cou
nt
100 101 102 103 1040
25
50
75
100
FL1 H
Count
100 101 102 103 1040
25
50
75
100
FL1-H
Count
100 101 102 103 1040
25
50
75
100
Raji
Raji 4RH
JeKo
Z138
UnstainedFITCCD20
Comparison of the CD20 in the rituximab sensitive Raji cells and
rituximab resitant raji cells with JeKo and Z138 MCL cell
lines.
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0102030405060708090
MINO JEKO Z138 REC-1 Raji Raji 4RH
CD20
Figure: comparing the CD 20 among the MCL cell lines and Raji
rituximab sensitive and rituximab resistant cells.
Chart2
MINO
JEKO
Z138
REC-1
Raji
Raji 4RH
CD20
CD20
67.98
63.56
53.73
81.36
42.98
15.61
Sheet1
Cell lineCD20CD55CD59
MINO67.98481.0323.74
JEKO63.56499.0812.98
Z13853.73318.6156.09
REC-181.36441.8759.2
Raji42.9885.722.59
Raji 4RH15.61526.6939.82
Sheet1
CD20
CD20
Sheet2
Sheet3
-
FL1 H
Coun
t
100 101 102 103 1040
25
50
75
100
Count
100 101 102 103 1040
25
50
75
100
FL1 H
Cou
nt
100 101 102 103 1040
25
50
75
100
FL1 H
Cou
nt
100 101 102 103 1040
25
50
75
100
UnstainedCD59FITC Isotype
JeKo REC-1
Z138 MINO
Surface expression of CD 59 in MCL cell lines
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4-21-2010 CD59 Raji RL.007
FL1-H
Coun
t
100 101 102 103 1040
41
83
124
1654-21-2010 CD59 Raji RL.001
FL1-H
Count
100 101 102 103 1040
25
50
75
100
FL1 H
Coun
t
100 101 102 103 1040
25
50
75
100
FL1 H
Count
100 101 102 103 1040
25
50
75
100
Raji
Raji 4RH
JeKo
Z-138
UnstainedCD59FITC Isotype
Comparison of the CD59 in the rituximab sensitive Raji cells and
rituximab resitant raji cells with JeKo and Z138 MCL cell
lines.
-
Figure: comparing the CD59 among the MCL cell lines and Raji
rituximab sensitive and rituximab resistant cells.
010203040506070
MINO JEKO Z138 REC-1 Raji Raji 4RH
CD59
Chart4
MINO
JEKO
Z138
REC-1
Raji
Raji 4RH
CD59
23.74
12.98
56.09
59.2
22.59
39.82
Sheet1
Cell lineCD20CD55CD59
MINO67.98481.0323.74
JEKO63.56499.0812.98
Z13853.73318.6156.09
REC-181.36441.8759.2
Raji42.9885.722.59
Raji 4RH15.61526.6939.82
Sheet1
CD20
CD20
Sheet2
CD55
Sheet3
CD59
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FL4-H
Coun
t
100 101 102 103 1040
25
50
75
100
Coun
t
100 101 102 103 1040
25
50
75
100
FL4-H
Cou
nt
100 101 102 103 1040
25
50
75
100
FL4 H
Coun
t
100 101 102 103 1040
25
50
75
100
UnstainedAPCC IsotypeCD55
JeKo REC-1
Z-138 MINO
Surface expression of CD 55 in MCL cell lines
-
FL4-H
Count
100 101 102 103 1040
25
50
75
100
FL4-H
Coun
t
100 101 102 103 1040
25
50
75
100
FL4-H
Coun
t
100 101 102 103 1040
25
50
75
100
Cou
nt
100 101 102 103 1040
25
50
75
100
Raji
Raji 4RH
JeKo
Z138
UnstainedAPCC IsotypeCD55
Comparison of the CD55 in the rituximab sensitive Raji cells and
rituximab resitant raji cells with JeKo and Z138 MCL cell
lines.
-
Figure: comparing the CD55 among the MCL cell lines and Raji
rituximab sensitive and rituximab resistant cells.
0100200300400500600
MINO JEKO Z138 REC-1 Raji Raji4RH
CD55
Chart3
MINO
JEKO
Z138
REC-1
Raji
Raji 4RH
CD55
481.03
499.08
318.61
441.87
85.7
526.69
Sheet1
Cell lineCD20CD55CD59
MINO67.98481.0323.74
JEKO63.56499.0812.98
Z13853.73318.6156.09
REC-181.36441.8759.2
Raji42.9885.722.59
Raji 4RH15.61526.6939.82
Sheet1
CD20
CD20
Sheet2
CD55
Sheet3
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CONCLUSION
• Ofatumumab and rituximab have comparable levels of cell lysis
in ADCC assays in all MCL cell lines.
• Ofatumumab induced significantly higher levels of cell lysis
compared to rituximab in CDC assays in all MCL cell lines
• Flow cytometry showed similar levels of CD 20 expression in
all the MCL cell lines and when compared with rituximab sensitive
Raji cells also.
• Complement inhibitory proteins particularly CD 55 were higher
and are comparable to rituxmab resistant Raji cells explaining the
difference between the activity of rituximab and ofatumumab.
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Further studies on the pre clinical activity of ofatumumab and
rituximab in MCL cell lines.
• In primary tumor cells, OFA and RTX demonstrated similar
activity.
• Expression of complement inhibitory proteins (CIPs) CD55 and
CD59 was determined by Imagestream analysis and Western blot.
• SCID mice were inoculated SQ with 10x106 Z-138 cells. Once
tumors were established, mice were assigned to observation versus 4
doses of either OFA or RTX, and anti-tumor activity was measured by
changes in tumor volume.
• Imagestream analysis and western blot techniques were used to
accurately delineate the surface expression of CD 20 and complement
inhibitory proteins.
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Dr. Matthew Barth MD Cory Mavis MS
Dr. Myron Cuczman MD Dr. Francisco J. Hernandez-Ilizaliturri
MD
THANK YOU
Cancer is a word, not a sentence. John Diamond
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