ACTIVITY OF RITUXIMAB AND OFATUMUMAB AGAINST MANTLE CELL … · •CDC with ofatumumab is not...

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

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

•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).


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


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.


•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

PB-Peripheral blood.

BM-Bone marrow.

Minimal residual disease quantification by RQ-PCR of 190 patients before, during and after induction.


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


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


“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

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.

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

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

• 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.

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

•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 ═

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.

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

CDC on MCL cell lines

Z-138

JeKo

REC-1

MINO

REC-1

Z138

JEKO

MINO

CDC on MCL cell lines

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

•Ofatumumab and rituximab showed comparable level of cell lysis in ADCC assays

Z-138 JeKo

REC-1 MINO

ADCC on MCL cell lines

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.

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


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


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


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

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


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


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


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

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.

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.

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

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42

ACTIVITY OF RITUXIMAB AND OFATUMUMAB AGAINST MANTLE CELL … · •CDC with ofatumumab is not...

Documents

Transcript of ACTIVITY OF RITUXIMAB AND OFATUMUMAB AGAINST MANTLE CELL … · •CDC with ofatumumab is not...