Activity of Rituximab and Ofatumumab Against Mantle

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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).

•Spectrum of variants from classic type to blastoid and pleomorphic types

Classic MCL Blastoid MCL Pleomorphic MCL

Small–medium-sized lymphocytes Rounded nuclei, finely dispersed Larger cells with irregular and with irregular nuclei and chromatin and inconspicuous pleomorphic nuclei and distinct inconspicuous nucleoli nucleoli small nuclei

•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 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.

RD according to MRD status in PB RD duration according to RD duration according to and/or BM after end of induction in MRD status assessed in the PB after MRD status assessed in the BM after MCL Younger and MCL Elderly induction treatment in both trials. induction treatment in both trials. patients

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 RD according to MRD status assessed in PB and/or BM within the first 12 months after ASCT in MCL BM during thefirst year of maintenance in MCL Younger patients. 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?”. 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, o incubated them for 2 hrs at 37 c,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 cells +media Ofatumumab cells +OFA+ Serum/PBMC

Rituximab cells +RIT+ Serum/PBMC

Herceptin cells +HER2+Serum/PBMC

Serum/PBMC cells +serum/PBMC +Media

Detergent 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

51 51 % lysis ═ [ Cr release from sample- Cr 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

Unstained FITC JeKo REC-1 CD20

100 100

75 75 50

50 Count

Count 25 25 0 0 1 2 3 4 0 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 FL1-H FL1 H

Z138 MINO 100 100

75 75

50 50 Count 25 Count 25 0 0 1 2 3 4 0 10 10 10 10 10 0 1 2 3 4 FL1-H 10 10 10 10 10 FL1 H

Histograms of the flow cytometry for CD20 surface expression. 100

75 Unstained FITC 50

Count Raji CD20 25

0 0 1 2 3 4 10010 10 10 10 10 FL1-H

75 Raji.031 Raji 4RH Comparison of the CD20 in the 50 Count rituximab sensitive Raji cells 25 and rituximab resitant raji cells 0 0 1 2 3 4 with JeKo and Z138 MCL cell 100 10 10 10 10 10 FL1-H lines. 75 Raji4RHP10-2.034

Count JeKo 25

0 0 1 2 3 4 10 10 10 10 10 100 FL1 H

Count Z138 25

0 0 1 2 3 4 10 10 10 10 10 FL1-H

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 Is oty pe

JeKo REC-1 100 100

75 75

50 50 Count Count

25 25

0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1 H

MINO Z138 100 100

75 75

50 50 Count Count 25 25

0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1 H FL1 H

100

75 Unstained 50 Count Raji 25 CD59

0 0 1 2 3 4 10 10 10 10 10 FITC Is oty pe FL1-H

4-21-2010 CD59 Raji RL.001 165 124 Comparison of the CD59 in the 83 Count Raji 4RH rituximab sensitive Raji cells 41 and rituximab resitant raji cells 0 0 1 2 3 4 10 10 10 10 10 with JeKo and Z138 MCL cell 100 FL1-H

4-21-2010 CD59 Raji RL.007 lines. 75

50 Count JeKo 25

0 0 1 2 3 4 10 10 10 10 10 100 FL1 H

75 50 Count 25 Z-138 0 0 1 2 3 4 10 10 10 10 10 FL1 H

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

Unstained A PCC Is oty pe CD55

REC-1 JeKo

100 100

75 75

50 50 Count Count 25 25

0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL4-H

MINO Z-138 100 100

75 75

50 50 Count Count 25 25 0 0 1 2 3 4 0 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 FL4-H FL4 H

100

75 Raji Unstained 50

Count A PCC Is oty pe 25 CD55

0 0 1 2 3 4 10 10 10 10 10 100 FL4-H

75 Comparison of the CD55 in 50 Count Raji 4RH the rituximab sensitive Raji 25 cells and rituximab resitant 0 0 1 2 3 4 10 10 10 10 10 raji cells with JeKo and 100 FL4-H

Z138 MCL cell lines. 75

Count JeKo

0 0 1 2 3 4 10 10 10 10 10 100 FL4-H

50 Count 25 Z138

0 0 1 2 3 4 10 10 10 10 10

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