scholarly journals Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1763-1763 ◽  
Author(s):  
Emma Vilventhraraja ◽  
Tetyana Klymenko ◽  
Jennifer Edelmann ◽  
John Gribben ◽  
Andrejs Ivanov
Keyword(s):  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Mitochondrial Function ◽  
Type I ◽  
Type Ii ◽  
Antibody Treatment ◽  
Respiratory Capacity ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies. The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function. In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced. We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines. Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3721-3721
Author(s):  
Gerhard Niederfellner ◽  
Olaf Mundigl ◽  
Alexander Lifke ◽  
Andreas Franke ◽  
Ute Baer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mechanisms Of Action ◽  
Type I ◽  
Type Ii ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3726-3726 ◽  
Author(s):  
Gerhard J Niederfellner ◽  
Alfred Lammens ◽  
Manfred Schwaiger ◽  
Guy Georges ◽  
Kornelius Wiechmann ◽  
...  
Keyword(s):  
Research Funding ◽  
Type I ◽  
The Novel ◽  
Type Ii ◽  
Employment Equity ◽  
Equity Ownership ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Point Mutagenesis ◽  
Cd20 Antibodies

Abstract Abstract 3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies. Disclosures: Niederfellner: Roche: Employment. Schwaiger:Roche: Employment. Georges:Roche: Employment. Wiechmann:Roche: Research Funding. Franke:Roche: Research Funding. Schaefer:Roche: Employment. Jenewein:Roche: Employment. Slootstra:Pepscan: Employment, Patents & Royalties. Moessner:Glycart: Employment, Equity Ownership, Patents & Royalties. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner:Roche: Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

A Novel FACS Assay for Absolute Quantification of CD20 on B-Cells and Determination of CD20 Occupancy in Whole Blood Samples Shows a Higher Binding Strength of Obinutuzumab (GA101) to CD20 on B-Lymphocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4611-4611
Author(s):  
Drifa Beroual ◽  
Danièle Boulay-Moine ◽  
Carole Badoil ◽  
Christian Klein ◽  
Maxime Moulard
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Whole Blood ◽  
Absolute Number ◽  
Type Ii ◽  
Human Mabs ◽  
Cd20 Antibody ◽  
The Absolute ◽  
Anti Cd20

Abstract Abstract 4611 Introduction. The absolute quantification of CD20 on malignant B-lymphocytes on patients who already received anti-CD20 MAbs is technically challenging, but may play a role in identifying patients who will respond to subsequent treatment or help define patients that are rituximab-refractory due to decreased expression of CD20. We have developed a new quantitative flow cytometry assay to scrutinize the absolute number of receptors on cell lines and B-lymphocytes in whole blood (WB) samples as well as target occupancy. This assay allows us to accurately determine the number of human MAbs bound per target cell on various cell subsets. CD20 density on B-lymphocytes in WB and binding properties of the type II CD20 antibody obinutuzumab (GA101) and the classical Type I CD20 antibody Rituximab were tested on samples from healthy volunteers. Acidic striping of bound human anti-CD20 MAbs was explored to enable the set up of a trustworthy CD20 occupancy assay. Methods. Binding of anti-CD20 mAbs rituximab (RTX) and GA101 to CD20 on normal and malignant B-cells was assessed using the Human IgG Calibrator (BioCytex, F) on EDTA anticoagulated WB samples and cell lines to determine the CD20 density. In parallel, the murine MAb (Clone B9E9) was tested using Cellquant Calibrator (BioCytex, F). CD20 density and apparent Kd, a relative measure of affinity, for each MAb was calculated from the data obtained with whole blood samples. Both the total number of CD20 molecules and the CD20 bound fraction were appraised by spiking the sample with anti-CD20 MAbs at saturation and acid striping. Results. The absolute number of anti-CD20 MAbs bound to CD20 on B-cells in WB samples was between 58,000 to 123,000 molecules per cell as determined by GA101 and RTX, respectively. The apparent CD20 quantitation on cell lines was approx. 2- to 3-fold lower for GA101 which is a characteristic and reported property of type II CD20 antibodies. The apparent Kd of GA101 (4.1 to 9.9 nM) was approximately 2-fold lower than that of RTX (10.5 to 22.7 nM) on several different lymphoma-derived cell lines (Daudi, Raji, Ramos and MEC-1). On B-lymphocytes from healthy volunteers the Kd were 1.2 and 3.5 nM for GA101 and RTX, respectively. Furthermore, CD20-bound GA101 could only be detached from its target CD20 using acid-striping assay at pH < 2.0 and appropriate ionic strength whereas RTX could be stripped at pH 2.5. Binding of anti-CD20 MAbs to acid-treated B-lymphocytes was found unaffected. Conclusion. The density of RTX bound to CD20 on B-lymphocytes in whole blood is similar to previous published data obtained from radioimmunoassays and using 125I-MAb (Teeling et al, Blood, 2006). In contrast the number of the Type II CD20 antibody GA101 bound to B-lymphocytes in whole blood was approximately half in comparison to RTX as previously reported (Moessner et al, Blood, 2010). The data further confirmed a higher apparent binding affinity of GA101 for CD20 with Kd values comparable to those reported previously using 125I-MAb. In summary, the new Human IgG Calibrator is a fast and accurate technique that can be used to evaluate the CD20 density and occupancy using human MAbs in complex matrix including whole blood. The test can be implemented in pharmacodynamic clinical trials and may allow for further understanding of complex pharacodynamic relationships. Disclosures: Beroual: BioCytex: Employment. Boulay-Moine:BioCytex: Employment. Badoil:BioCytex: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Moulard:BioCytex: Consultancy, Employment.

Protein Tomography Analysis Shows That CD20 Molecules Bound by Different Type I and Type II Anti-CD20 Antibodies Are Organized in Different Protein Complexes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4782-4782
Author(s):  
Gerhard J Niederfellner ◽  
Annika Braennstroem ◽  
Frida Lindstrom ◽  
Magnus Jansson ◽  
Marika Lundin ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Large Fraction ◽  
Type I ◽  
Type Ii ◽  
Employment Equity ◽  
Downstream Signaling ◽  
Equity Ownership ◽  
Anti Cd20 ◽  
Tomography Analysis ◽  
Cd20 Antibodies

Abstract Abstract 4782 Ramos cells were labeled with Type I (Rituximab and Ocrelizumab) and Type II (B1/H299 and GA101) anti-CD20 antibodies and then fixed and stained with marker gold labeled 2ary antibodies. Protein complexes connected to marker gold were analyzed by Protein TomographyTM at SIDEC and the corresponding 3D-structures visualized (∼100 structures per antibody). Less than 5 % of the refined structures were dimeric. While most (60 – 90%) CD20 molecules were present in tetramers or even higher order defined complexes, a sizeable proportion was also engaged in large protein networks (11 -38%). For the multimeric complexes, we could clearly distinguish between extended (or open) and ring-like (or closed) conformations. Although cells had been labeled with an excess of antibody, as suggested by FACS binding curves, the antibodies bound CD20 monovalently in most refined structures. Bivalent binding was overall more prevalent with Rituximab and Ocrelizumab than with B1 and GA101 (1/3 vs 1/6 structures). The proportion of CD20 molecules present in ringlike complexes was higher for the Type II than for the Type I antibodies. In co-localization experiments, Rituximab-CD20 and GA101-CD20 complexes were also found to only partially colocalize, while a large fraction of the two antibodies were found in separate cell surface compartments. These findings suggest that the different antibodies favor different CD20 conformations that seem to be associated with different protein complexes and might form the basis for initiation of different downstream signaling processes. Disclosures: Niederfellner: Roche: Employment. Braennstroem:SIDEC: Employment. Lindstrom:SIDEC: Employment. Jansson:SIDEC: Employment. Lundin:SIDEC: Employment. Schaefer:Roche: Employment. Mundigl:Roche: Employment. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Superior Efficacy of the Novel Type II, Glycoengineered CD20 Antibody GA101vs. the Type I CD20 Antibodies Rituximab and Ofatumumab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3925-3925 ◽  
Author(s):  
Sylvia Herter ◽  
Inja Waldhauer ◽  
Tina Otz ◽  
Frank Herting ◽  
Sabine Lang ◽  
...  
Keyword(s):  
Cell Death ◽  
Type I ◽  
Type Ii ◽  
B Cell Depletion ◽  
Employment Equity ◽  
Direct Cell ◽  
Equity Ownership ◽  
Cd20 Antibody ◽  
Dose Dependent ◽  
Cd20 Antibodies

Abstract Abstract 3925 GA101 is Type II, glycoengineered CD20 monoclonal antibody currently in PhII/III clinical trials. We have previously shown that GA101 mediates superior in vitro and in vivo activity compared to the Type I CD20 antibody rituximab. By epitope mapping and crystallography we have shown that GA101 recognizes CD20 in a unique way that is different from Type I CD20 antibodies and have proposed that this may be the basis for the Type II character of GA101. Here we compare for the first time GA101 with rituximab, the standard of care in various clinical settings in NHL and B-CLL in combination with chemotherapy, as well as with the Type I CD20 antibody ofatumumab, which was recently approved for treatment of B-CLL patients refractory to fludarabine and alemtuzumab. The following assays were used to compare the three anti-CD20 antibodies: i) Binding to NHL cell lines Z138 (MCL, ca. 60.000 CD20 binding sites per cell) and SU-DHL4 (DLBCL, ca. 1 Mio CD20 binding sites per cell) assessed by FACS, ii) Cell death induction, detected by AxV/PI staining and FACS, on a panel of NHL cell lines, iii) Antibody dependent cellular cytotoxicity mediated by PBMNCs as effector and Z138, SU-DHL4 as target cells (ADCC, LDH release assay); iv) Complement dependent cytotoxicity with Z138, SU-DHL4 as target cells (CDC, LDH release assay) and v) B-cell depletion (assessed by FACS) in whole blood from healthy donors. Dose-dependent anti-tumoral activity was assessed in a s.c. SU-DHL4 NHL xenograft model in Scid beige mice. Survival experiments in a disseminated Z138 MCL model are ongoing and an update on the results will be included as part of the poster presentation. Ofatumumab (“Arzerra”) was purchased from a local pharmacy, GA101 and rituximab were obtained from Hoffmann La Roche AG, Basel. First, binding studies confirmed that GA101 shows half-maximal binding to NHL cells relative to rituximab and ofatumumab, a known property of Type II CD20 antibodies. EC50 values of binding were comparable indicating that GA101, rituximab and ofatumumab have apparent binding affinities in the low nanomolar range on NHL cells independent of the level of CD20 expression. Second, the three CD20 antibodies were compared for their induction of direct cell death as measured by AxV/PI staining. Overall, GA101 mediated superior direct cell death induction compared to rituximab and ofatumumab utilizing a panel of NHL cell lines of different origins. Immune effector-related mechanisms of action were subsequently compared by ADCC and CDC assays. GA101, a glycoengineered antibody with enhanced affinity for FcgRIIIa, was found to exhibit up to 100-fold higher ADCC potency than rituximab and ofatumumab on Z138 and SU-DHL4 cells. CDC, as expected for a Type II CD20 antibody was ca. 10 to 1,000 less potent compared to the Type I antibodies rituximab and ofatumumab. In order to integrate the different mechanisms of action (direct cell death, ADCC, CDC), autologous ex vivo B-cell depletion assays with whole blood from healthy donors containing natural immune effector cells, human complement and physiological concentrations of human immunoglobulins were performed. These studies showed that GA101 was more potent in terms of EC50 values and more efficacious in terms of absolute B-cell depletion when compared to rituximab and ofatumumab. Finally, the dose-dependent effects of the three CD20 antibodies was studied on the growth of s.c. SU-DHL4 DLBCL xenografts in SCID beige mice. GA101 induced a dose-dependent anti-tumoral effect including complete tumor remission and was superior to the Type I antibodies rituximab and ofatumumab at saturating antibody doses. In summary, the preclinical data presented herein demonstrate that the Type II, glycoengineered CD20 antibody GA101 is differentiated from the Type I CD20 antibodies rituximab and ofatumumab by its superior overall activity supporting its further clinical investigation. Of note, in contrast to previous publications, in this series of assays no superior preclinical activity of ofatumumab was observed when compared to rituximab. Disclosures: Herter: Roche: Employment, Patents & Royalties. Waldhauer:Roche: Employment. Otz:Roche: Employment. Herting:Roche: Employment, Patents & Royalties. Lang:Roche: Employment. Nicolini:Roche: Employment. Römmele:Roche: Employment. Friess:Roche: Employment, Patents & Royalties. Van Puijenbroek:Roche: Employment. Bacac:Roche: Employment. Weidner:Roche: Employment, Equity Ownership. Gerdes:Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

scholarly journals CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4174-4174
Author(s):  
Ravi Dashnamoorthy ◽  
Afshin Beheshti ◽  
Saheli Sarkar ◽  
Pooja Sabhachandani ◽  
Frank C. Passero ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Resistant Cell ◽  
Cytolytic Activity ◽  
Lymphoma Cells ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Targeted Immunotherapy ◽  
Cd20 Antibodies

Abstract Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines. Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment. Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells. Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. Figure Expression of NK activation and inhibitory ligands in lymphoma cells. Figure. Expression of NK activation and inhibitory ligands in lymphoma cells. Disclosures Boissel: NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

Enhanced Killing of Human B Lymphoma Targets by Combined Use of Cytokine Induced Killer (CIK) Cultures and Anti-CD20 Antibodies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4285-4285
Author(s):  
Alice Pievani ◽  
Camilla Belussi ◽  
Christian Klein ◽  
Alessandro Rambaldi ◽  
Josée Golay ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cell ◽  
Type I ◽  
Type Ii ◽  
B Lymphoma ◽  
Cik Cells ◽  
High Concentrations ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Abstract 4285 Cytokine induced killer (CIK) cells are immune-effector cells that can be expanded in vitro in presence of rhIL-2, starting from peripheral blood mononuclear cells stimulated by interferon-γ and anti-CD3 antibody. CIK cultures at the end of in vitro expansion contain a mean of 40–75% CD3+CD56+ CIK cells, 20–60% CD3+CD56- T cells and 1–10% CD3-CD56+ NK cells. They show MHC-unrestricted cytotoxicity towards neoplastic but not normal targets. Their ease of production in vitro and anti-tumor potential have made them suitable candidates for cell therapy programs in solid and hematopoietic tumour treatment. CIK cells have shown cytotoxic activity in vitro against hematopoietic neoplastic cells, including B Non-Hodgkin's lymphoma (B-NHL). Other biological treatments available for B-NHL are the anti-CD20 antibodies such as type I Rituximab and a new generation glycoengineered type II GA101 antibody. These antibodies are thought to act mostly through immune mediated mechanisms including phagocytosis, complement mediated cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). GA101 has reduced CDC activity compared to type I anti-CD20 antibodies such as Rituximab. In addition, GA101 was defucosylated in its Fc portion to mediate increased ADCC. We have investigated the possibility of combining adoptive immunotherapy by Cytokine Induced Killer (CIK) cells with anti-CD20 type I Rituximab and type II GA101mAb, to optimize B-NHL therapy. CIK cultures alone demonstrated significant cytotoxic activity against a panel of B-NHL cell lines or freshly isolated samples, in either an autologous or allogeneic combination (26-27% killing at 30:1 ET ratio). This natural cytotoxic activity was mainly due to the predominating CD3+CD56+ CIK population (40-75%) present in the cultures. The addition of anti-CD20 mAbs increased CIK mediated cytotoxicity versus B lymphoma target cells and major enhancement was observed with GA101 compared to Rituximab (respectively 34% versus 16% increased lysis at 10:1 E:T ratio). This enhancement was mainly due to ADCC mediated by the small NK cell fraction (1-10%) present in CIK cultures because NK depletion by CD5 immunoselection at the end of expansion did not abolish the basal natural cytotoxicity of CIK cultures but abolished the enhancement observed in presence of anti-CD20 antibodies. The activation of NK cells in CIK cultures, evaluated by CD107a mobilization, was much more effective using GA101 rather than Rituximab (respectively 28% versus 19% CD107a+, p<0.005). Furthermore, in presence of human serum, Rituximab-mediated NK cell activation was inhibited to 70%, whereas the GA101-mediated was fully maintained. This inhibition was presumably due to complement C3 deposition, since it was not observed with either heat inactivated serum or high concentrations of human immunoglobulins. Inhibition was however observed with serum plus anti-C5 antibody eculizumab, which blocks the complement cascade downstream from C3. Lack of inhibition by serum of GA101-mediated NK cell degranulation was probably due to the higher affinity binding of this mAb to CD16 and not to a lower C3 activation, because at high concentrations, GA101 is nearly as efficient as Rituximab at activating complement and C3 deposition. Finally, addition of serum to CIK and mAbs did not modify overall the target cell killing by either antibody. More importantly, the use of a partially complement and CIK resistant lymphoma cell line such as WSU-NHL showed that resistance could be reverted by combined exposure to CIK cultures and monoclonal anti-CD20 antibodies. Indeed overall lysis of WSU-NHL, even in presence of serum, was significantly increasedby both anti-CD20 antibodies, and most effectively by GA101, compared to CIK cells alone. This was due to the combined action of CDC, ADCC and CIK mediated natural killing. In conclusion, CIK cultures could be used to treat B-NHL patients in both an autologous or allogeneic setting. Furthermore Rituximab but even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL. These data may open the way to possible therapeutic exploitation of combined strategies of cell mediated and antibody mediated immunotherapy protocols which make use of different mechanisms of action to try and overcome resistance. Disclosures: Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Rambaldi:Roche: Honoraria. Golay:Roche: Honoraria. Introna:Roche: Honoraria.

A Phase I/II Study of RO5072759 (GA101) in Patients with Relapsed/Refractory CD20+ Malignant Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 234-234 ◽  
Author(s):  
Gilles Andre Salles ◽  
Franck Morschhauser ◽  
Guillaume Cartron ◽  
Thierry Lamy ◽  
Noel-Jean Milpied ◽  
...  
Keyword(s):  
Phase I ◽  
Malignant Disease ◽  
Single Agent ◽  
Tumor Lysis Syndrome ◽  
T Cell Subsets ◽  
Type I ◽  
Type Ii ◽  
Cell Counts ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract RO5072759 (GA101) is the first humanized and glycoengineered monoclonal anti-CD20 antibody to enter clinical trials. Glycoengineeering results in a significantly increased antibody-dependent cytotoxicity (ADCC) compared to rituximab as shown in in vitro models. Additionally, RO5072759 binds with high affinity to a type II epitope on CD20 and is characterized by reduced complement-dependent cytotoxicity (CDC) and strongly enhanced direct cell death compared to type I antibodies. In preclinical in vivo lymphoma models RO5072759 has shown superior efficacy compared to rituximab. In this Phase I/IIa study RO5072759 was administered as a single agent to patients with CD20+ malignant disease for whom no therapy of higher priority was available. The aim of which was to determine the safety and tolerability of RO5072759, any dose-limiting toxicity (DLT), its pharmacokinetics and to establish the recommended phase II dose. Patients were treated with RO5072759 by intravenous infusion (premedication with acetaminophen and anti-histamines) administered as a flat dose on days 1, 8 and 22 and subsequently every 3 weeks for a total of 9 infusions. The dose of the first infusion was 50% that of subsequent infusions. The dose was escalated based on the safety in a 3+3 design. Since September 2007 twenty four patients have been treated with RO5072759 at doses from 50 mg to 2000 mg. Complete data, presented herein, is available on the first 12 patients, median age 59 yrs (39–83), from the first 4 cohorts (50 mg–800 mg). Most patients had follicular NHL (9) others include DLBCL (1) CLL (1) Waldenstrom’s macroglobulinemia (1). All were previously exposed to rituximab and had received a median of 4 (range 1–7) prior regimens. RO5072759 was well tolerated with no DLTs observed. The most common adverse events are Grade 1 or 2 (CTCAE V3.0) infusion related reactions, characterized by fever, chills, hypo/hypertension, nausea and vomiting limited in the main, to the first infusion. These responded well to slowing or interruption (5 pts) of the infusion and steroids (1 pt). No tumor lysis syndrome has been observed. Only 6 minor infections (4 upper respiratory track, 1 urinary track and 1 oral herpes) were observed to-date. Measurement of plasma cytokines and complement during and immediately after the first infusion showed an increase in IL6 and IL8 with a smaller increase in IL10 and TNFα. No change in complement fractions (C3, C3a, C4a, C5, C5a, Bb) was observed. Concurrent to cytokine increase was apparent a decrease in T-cell subsets (CD3, CD4 and CD8 subsets) and NK cell counts in the peripheral blood. This decrease, as with the cytokine increase, was transient and levels were at or near baseline by day 8. No similar changes, for the majority of patients, were seen with subsequent infusions. Circulating B-cell (CD19) depletion occurs rapidly and is sustained. The pharmacokinetics of RO5072759 are broadly similar to those of rituximab and show a dose-dependent increase in exposure, but with significant inter- and intra-patient variability. Time dependent clearance was noted which is consistent with a reduction in target-mediated antibody clearance with increasing duration of treatment. To-date 7 of 12 patients have responded by day 85 to treatment [3 CR (25%), 4 PR (33%) ORR (58%)], 1 patient has SD at 8 months, 3 patients have PD (2 have since died) and 1 patient died from an event unrelated to treatment (cerebrovascular accident). Responses have occurred at all dose levels. In conclusion, RO5072759 is a novel ADCC-enhanced type II anti-CD20 antibody that has shown a similar safety profile to rituximab and promising efficacy in this difficult-to-treat patient population. Dose finding is continuing and data from all 24 patients will be presented.

Novel 3rd Generation Humanized Type II CD20 Antibody with Glycoengineered Fc and Modified Elbow Hinge for Enhanced ADCC and Superior Apoptosis Induction.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 229-229 ◽  
Author(s):  
Pablo Umana ◽  
Ekkehard Moessner ◽  
Peter Bruenker ◽  
Gabriele Unsin ◽  
Ursula Puentener ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Third Generation ◽  
Type Ii ◽  
Apoptosis Induction ◽  
B Cell Malignancies ◽  
Cd20 Antibody ◽  
Cd20 Antibodies

Abstract Background: Treatment of B-cell non-Hodgkin lymphoma (NHL) with antibodies targeting CD20 in conjunction with combination chemotherapy is standard clinical practice. Two different types of CD20 MAb differing significantly in their mode of CD20 binding and biological activities have been identified (Cragg and Glennie. Blood103: 2738–2743, 2004): type I antibodies, as rituximab, are potent in complement mediated cytotoxicity, whereas type II antibodies, as tositumomab, effectively initiate target cell death via caspase-independent apoptosis with concomitant phosphatidylserine exposure. GA101 is a humanized and optimized, third generation, type II CD20 IgG1 antibody that exhibits enhanced ADCC and superior caspase-independent apotosis induction in comparison with currently available CD20 MAbs. Material and Methods: GA101 was humanized by grafting CDR sequences from the murine monoclonal antibody B-ly1 on framework regions with fully human IgG1-kappa germline sequences. During humanization different elbow hinge sequences in the variable region were studied for their capability to induce apoptosis. Furthermore, the Fc region-carbohydrates were glycoengineered using GlycoMAb™ technology leading to bisected, afucosylated Fc region-carbohydrates. Results: The humanized GA101 antibody bound CD20 as type II antibody with nanomolar affinity. Its glycoengineered Fc region bound with 50-fold higher affinity to human FcgammaRIII receptors compared to a standard, non-glycoengineered antibody. Increased FcgammaRIII binding led to a 10–100-fold increase in ADCC against CD20-expressing NHL cell lines. Modification of elbow hinge sequences within the antibody variable framework regions resulted in a strong apoptosis-inducing activity of GA101 upon CD20 binding on target cells. Direct comparison to other CD20 antibodies GA101 showed enhanced apoptosis induction in both a panel of NHL cell lines and ex vivo in samples from patients with a variety of B-cell malignancies. Furthermore, in B-cell depletion assays with whole blood from healthy donors and B-cell leukemic patients, an assay combining ADCC-, CDC- and apoptosis-mediated mechanisms of action, GA101 was significantly more potent and efficacious than other CD20 antibodies, including rituximab and Fc-variants of rituximab that have increased ADCC. Finally, the in vitro superiority of GA101 also translated into superior efficacy in vivo. In NHL xenograft models of different histological origin, including aggressive DLBCL and MCL, treatment with GA101 results in complete tumor remission and long-term survival (cure) compared to tumor stasis, at best, for rituximab. Conclusion: Compared to existing CD20 antibodies GA101 represents a novel, third generation antibody with significantly enhanced efficacy in a variety of in vitro and in vivo preclinical models. GA101 constitutes the first type II CD20 antibody successfully engineered for increased ADCC. Based on these data, GA101 is a promising therapeutic antibody candidate for the treatment of B-cell malignancies.

Target cell killing effects of CD20 targeting chimeric antigen receptor T cells derived from the type II anti-CD20 antibody.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14548-e14548 ◽  
Author(s):  
Yihong Yao ◽  
Xin Yao ◽  
Shigui Zhu ◽  
Wei Zhu ◽  
Zhiyuan Li ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Type I ◽  
Type Ii ◽  
Hydrophobic Residues ◽  
Car T ◽  
Small Loop ◽  
Cd20 Antibody ◽  
Anti Cd20

e14548 Background: Chimeric Antigen Receptor T cells (CAR-Ts) targeting CD19 have shown very promising clinical outcomes in treatment of B-cell linage hematological malignancies. However, many patients with relapsed diseases were found to have down-regulated/loss of CD19 surface expression after CD19 CAR-T therapy. To solve this issue of CD19 single-targeting escape, we explored the application of another B-cell antigen, CD20, for targeted CAR-T therapy. Methods: We constructed four CD20 targeting CARs (all with 4-1BB co-stimulatory signaling) base on single-chain variable fragments (scFV) derived from four well-studied CD20 specific antibodies: Leu16, Rituximab, Obinutuzumab, and Ofatumumab. Leu16, Rituximab, and Obinutuzumab belong to the type I anti-CD20 antibody family and appear to bind to different epitopes located on the large loop of CD20, whereas Ofatumumab is the type II anti-CD20 antibody which has been shown to interact with the hydrophobic residues on the small loop surrounding a deep binding cleft. Results: All four CAR-T cells can specifically recognized CD20 positive target cells in our pre-clinical studies. They all showed up-regulated antigen-specific cell activation and high level of IFN-g release upon CD20 stimulation, and CAR-T20-Ofatumumab cells appeared to have significantly higher cell activation and more than 2-fold increase in IFN-g release compared to the other three CAR-T20 cells with their scFVs deriving from type I anti-CD20 antibodies. CAR-T20-Ofatumumab cells also showed higher degranulation upon stimulation, and it displayed ~50% of increase in ability to kill CD20 positive cells in cytotoxicity assays. Conclusions: Our data suggested that CAR-T20-Ofatumumab has better in vitro function and appears to be a CAR superior to those derived from other three antibodies. A possible explanation for this observation is that Ofatumumab interacts with the hydrophobic residues on the small loop, which is very close to cell membrane and confers more extensive binding to the small loop with striking slow off-rate. Our results suggest that CAR-Ts targeting CD20 with the scFVs from the type II anti-CD20 antibody may have superior cell killing effects.

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