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)
Dr. Gopichand Pendurti M.B.B.S
Mentor: Dr. Francisco J. Hernandez-Ilizaliturri MD
Overview of presentation
•Introduction to mantle cell lymphoma.
•Concept of minimal residual disease.
•Anti CD 20 antibodies.
•51Cr release assays.
•Flow cytometry on cell lines.
•Results.
•Future.
MANTLE CELL LYMPHOMA
•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.
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
•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
MORPHOLOGY
•Spectrum of variants from classic type to blastoid and pleomorphic types
Classic MCL
Small–medium-sized lymphocytes
with irregular nuclei and
inconspicuous nucleoli
Blastoid MCL
Rounded nuclei, finely dispersed
chromatin and inconspicuous
nucleoli
Pleomorphic MCL
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-cellassociated 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
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
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
RESEARCH QUESTION
“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?”.
Edwards et al. Nature Reviews Immunology 6, 394–403 (May 2006) | doi:10.1038/nri1838
Mechanisms of Action of Anti-CD20 Antibodies
Maloney DG. N Engl J Med 2012;366:2008-2016.
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.
Binding site of Ofatumumab to CD 20 on the B-cells
Arzerra:Ofatumumab
The binding of ARZERRA to CD20. (2011). Retrieved May 9th from http://hcp.gsk.com/therapy_areas/oncology/arzerra/mechanism-of-action/#R4
Ofatumumab induces cell lysis by CDC
Arzerra:Ofatumumab
ARZERRA induces cell lysis by CDC. (2011). Retrieved on May 9th from http://hcp.gsk.com/therapy_areas/oncology/arzerra/mechanism-of-action/#R4
• 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.
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.
•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.
Media
Ofatumumab
Rituximab
Herceptin
Serum/PBMC
Detergent
cells +media
cells +OFA+ Serum/PBMC
cells +RIT+ Serum/PBMC
cells +HER2+Serum/PBMC
cells +serum/PBMC +Media
cells +media+ detergent
Model of the 96 well plate prepared for the experiments
•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
% lysis ═
[51Cr release from sample-51Cr release from control]
*100
[51Cr release from maximum release-51Cr release from control]
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.
STATISTICS
• SPSS 16 was used for the independent t-Test to calculate the
significance of the lysis between the two anti CD 20 antibodies.
RESULTS
•Ofatumumab induced significantly higher levels of cell lysis compared to
rituximab in CDC assays.
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%
p-value significant at <0.001
CDC on MCL cell lines
REC-1
Z-138
JeKo
MINO
CDC on MCL cell lines
REC-1
Z138
JEKO
MINO
•Ofatumumab and rituximab have comparable levels of cell lysis in ADCC
assays.
MCL Cell line
Ofatumumab
Rituximab
REC-1
12%
14%
Z-138
14%
12%
Mino
1%
3%
JeKo
12%
12%
p value not significant - 0.264
•Ofatumumab and rituximab showed comparable level of cell lysis in ADCC
assays
Z-138
JeKo
REC-1
MINO
ADCC on MCL cell lines
Surface expression of CD 20 in MCL cell lines
100
Count
75
Unstained
FITC
CD20
50
JeKo
REC-1
25
0
100
100
0
1
50
2
75
10
Count
10
75
3
10
4
10
10
FL1-H
50
25
jeko cells.001
25
0
0
0
1
10
0
10
1
10
2
10
3
10
10
4
2
10
3
10
4
10
FL1-H
10
FL1-H
reco cells.007
jeko cells.001
Z138
MINO
100
75
75
Count
100
50
25
50
25
0
0
10
1
10
2
10
3
10
4
10
FL1-H
0
0
10
1
10
2
10
3
10
4
10
FL1-H
z138 cells .013
Mino blank.001
Histograms of the flow cytometry for CD20 surface expression.
100
Unstained
FITC
CD20
Count
75
Raji
50
25
0
1
100100
1
10
10
2
10
2
10
3
10
FL1-H
FL1-H
75
3
10
4
10
4
10
Raji.031
Count
Raji 4RH
jeko cells.001
50
25
0
0
1
100 10
10
2
10
3
10
FL1-H
Count
75
Count
4
10
Raji4RHP10-2.034
JeKo
50
25
0
0
10
1
10
2
10
3
10
4
10
FL1-H
100
75
jeko cells.001
50
Z138
25
0
0
10
1
10
2
10
FL1-H
3
10
4
10
Comparison of the CD20 in the
rituximab sensitive Raji cells
and rituximab resitant raji cells
with JeKo and Z138 MCL cell
lines.
CD20
90
80
70
60
50
40
30
20
10
0
MINO
JEKO
Z138
REC-1
Raji
Raji 4RH
Figure: comparing the CD 20 among the MCL cell lines and Raji
rituximab sensitive and rituximab resistant cells.
Surface expression of CD 59 in MCL cell lines
Unstained
CD59
FITC Isotype
JeKo
REC-1
100
100
Unstained
CD59 75
FITC Isotype
Count
75
50
50
25
25
0
0
0
10
1
2
10
1
10
3
2
10
FL1-H
10
10
4
10
0
10
3
1
10
10
2
4
10
jeko cells.001
FL1-H
Z138
10
3
10
4
10
FL1-H
reco cells.007
MINO
jeko cells.001
100
75
75
Count
100
50
50
25
25
0
0
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
FL1-H
FL1-H
z138 cells .013
Mino blank.001
-H
100
75
Raji
50
25
0
0
10
1
10
2
10
3
10
4
10
Unstained
CD59
FITC Isotype
FL1-H
4-21-2010 CD59 Raji RL.001
165
124
Raji 4RH
83
41
0
0
10
1
10
2
10
3
10
4
10
FL1-H
100
Unstained
CD59
FITC Isotype
4-21-2010 CD59 Raji RL.007
75
50
3
4
JeKo
10
10
25
0
0
1
10
10
100
2
10
3
10
4
10
FL1-H
jeko cells.001
jeko cells.001
75
50
Z-138
25
0
0
10
1
10
2
10
3
10
4
10
FL1-H
z138 cells .013
Comparison of the CD59 in the
rituximab sensitive Raji cells
and rituximab resitant raji cells
with JeKo and Z138 MCL cell
lines.
CD59
70
60
50
40
30
20
10
0
MINO
JEKO
Z138
REC-1
Raji
Raji 4RH
Figure: comparing the CD59 among the MCL cell lines and
Raji rituximab sensitive and rituximab resistant cells.
Surface expression of CD 55 in MCL cell lines
100
Unstained
APCC Isotype
CD55
75
50
REC-1
JeKo
25
100
100
75
0
50
10
0
1
Count
75
2
10
10
3
FL4-H
25
4
10 50
10
25
0
0
0
10
1
10
2
10
3
10
0
10
4
10
1
10
jeko cells.001
FL4-H
2
10
3
10
4
10
FL4-H
jeko cells.001
MINO
Z-138
reco cells.007
100
75
75
Count
100
50
50
25
25
0
0
0
0
10
1
10
2
10
3
10
4
10
10
1
10
2
10
3
10
4
10
FL4-H
FL4-H
z138 cells .013
100
100
75
Unstained
APCC Isotype
CD55
Raji
75
50
25
50
0
0
10
100
1
10
2
10
3
4
10
10
FL4-H
4-28-10 CD55 Raji RL.001
75
25
50
Raji 4RH
25
00
0
10
100
0
10
1
10
2
110
10FL4-H
3
2
10
10
4
3
10
10
Unstained
FL4-H
4-28-10 CD55 Raji RL.004
75
APCC Isotype
CD55
50
JeKo
25
0
0
10
100
1
10
2
10
3
10
4
10
FL4-H
jeko cells.001
75
50
Z138
25
0
0
10
1
10
2
10
3
10
Comparison of the CD55 in
the rituximab sensitive Raji
cells and rituximab resitant
raji cells
4 with JeKo and
Z13810MCL cell lines.
4
10
FL4-H
z138 cells .013
jeko cells.001
CD55
600
500
400
300
200
100
0
MINO
JEKO
Z138
REC-1
Raji
Raji
4RH
Figure: comparing the CD55 among the MCL cell lines and
Raji rituximab sensitive and rituximab resistant cells.
CONCLUSION
• Ofatumumab induced significantly higher levels of cell lysis compared to
rituximab in CDC assays in all MCL cell lines
• Ofatumumab and rituximab have comparable levels of cell lysis in ADCC
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.
Further studies on the pre clinical activity of
ofatumumab and rituximab in MCL cell lines.
• Imagestream analysis and western blot techniques were used to
accurately delineate the surface expression of CD 20 and
complement inhibitory proteins.
• Expression of complement inhibitory proteins (CIPs) CD55 and CD59
was determined by Imagestream analysis and Western blot.
• In primary tumor cells, OFA and RTX demonstrated similar activity.
• 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.
THANK YOU
Dr. Matthew Barth MD
Dr. Myron Cuczman MD
Cory Mavis MS
Dr. Francisco J. Hernandez-Ilizaliturri MD
Cancer is a word, not a sentence.
John Diamond