We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.

BackgroundRituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.Methods and FindingsComplement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.ConclusionsStatins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

SummaryWe evaluated the dose requirements for sustained in vivo activity of ofatumumab, a human anti‐CD20 antibody under development for the treatment of B cell‐mediated diseases. In a mouse xenograft model, a single dose, resulting in an initial plasma antibody concentration of 5 μg/ml, which was expected to result in full target saturation, effectively inhibited human B‐cell tumour development. Tumour growth resumed when plasma concentrations dropped below levels that are expected to result in half‐maximal saturation. Notably, tumour load significantly impacted antibody pharmacokinetics. In monkeys, initial depletion of circulating and tissue residing B cells required relatively high‐dose levels. Re‐population of B‐cell compartments, however, only became detectable when ofatumumab levels dropped below 10 μg/ml. We conclude that, once saturation of CD20 throughout the body has been reached by high initial dosing, plasma concentrations that maintain target saturation on circulating cells (5–10 μg/ml) are probably sufficient for sustained biological activity. These observations may provide a rationale for establishing dosing schedules for maintenance immunotherapy following initial depletion of CD20 positive (tumour) cells.

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20 B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab') fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20 cells: OFA-F(ab') fragments were able to induce CDC in the CD20BCR cell population, but not in the CD20BCR population. Importantly, OFA-F(ab') fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.

The online version of this article has a Supplementary Appendix. BackgroundIncorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin's lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule. Design and MethodsBy retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells. ResultsWe show that HuMab-7D8 efficiently killed CD20 low cells that are not susceptible to rituximabinduced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20 low cells survived. ConclusionsCells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic. © F e r r a t a S t o r t i F o u n d a t i o n

1725 Poster Board I-751 Introduction CD20 represents a well established target for immunotherapy of B-cell malignancies such as follicular lymphoma (FL). The standard therapy for FL is the monoclonal antibody (mAb) rituximab given as a single agent or combined with chemotherapy. Antibody-dependent cytotoxicity (ADCC) has been suggested to be an important in vivo mechanism of action of CD20 mAb as differences in response rates to rituximab exist between individuals expressing different allotypes of the polymorphic Fc receptor FcgRIIIa. Individuals homozygous for the FcgRIIIa 158V allotype respond significantly better than individuals expressing the FcgRIIIa 158F allotype. Ofatumumab, a unique human mAb targeting a novel membrane-proximal small-loop epitope on CD20, has been shown to be associated with highly efficient killing of primary tumor cells through complement-dependent cytotoxicity. Here we studied the ability of ofatumumab to induce ADCC by NK cells from FcgRIIIa 158V and 158F homozygous healthy donors and compared it to that of rituximab. Methods Blood was drawn (with informed consent) from ten healthy volunteers homozygous for FcgRIIIa 158V, and ten homozygous for FcgRIIIa 158F, selected from a panel of 479 donors and matched for age, sex and race. NK cells were purified, after which we assessed Fc-mediated antibody binding to NK cells, and ADCC, in a blinded study. Glycan profiling was performed for ofatumumab and rituximab by HPAEC-PAD analysis. MAb binding was measured by flow cytometry in a competition binding assay in which binding of FITC-labelled CD16 mAb 3G8 was blocked by the binding of ofatumumab or rituximab. ADCC was assessed by measuring Europium release from the B cell line ARH77 target cells upon incubation with a concentration curve of ofatumumab or rituximab in the presence of NK effector cells (effector:target ratio 5:1). Statistical analysis was performed using a non-linear mixed effect model fitting sigmoidal concentration-responses curves to data from each donor for each antibody. The model provided estimates of mean EC50 for each antibody and donor group, and allowed differences between these means to be tested for statistical significance. Results Biochemical analysis of clinical grade ofatumumab and rituximab batches indicated their quality to be comparable with similar amounts of aggregates present. The level of core-fucosylation was determined to be similar and ranged from 4 – 6 % for both mAbs. Monomeric ofatumumab bound more strongly to NK cells expressing FcgRIIIa 158 V/V (EC50: 900 mg/ml [95% CI 680-1200 mg/ml]) than FcgRIIIa 158 F/F (EC50: 3970 mg/ml [95% CI 2940-5370 mg/ml]). The approximately 4.4 fold difference in affinity was statistically significant (p<0.0001). A similar 4.2 fold difference (p<0.0001) was found for rituximab (FcgRIIIa 158 V/V EC50: 1370 mg/ml [95% CI 1020-1850 mg/ml] and FcgRIIIa 158 F/F EC50: 5720 mg/ml [95% CI 4170-7850 mg/ml]). Rituximab bound 1.4 fold less tightly to both FcgRIIIa allotypes than ofatumumab (p<0.0001). Ofatumumab induced a potent NK-mediated ADCC with both FcgRIIIa 158V/V expressing (EC50: 6.4 ng/ml [95% CI 4.8-8.5 ng/ml]) as well as FcgRIIIa 158 F/F expressing NK cells (EC50: 17.6 ng/ml [95% CI 13.4-23.1 ng/ml]). The observed ∼2.7 fold difference in potency was significant (p< 0.0001). Notably, higher concentrations of rituximab compared to ofatumumab were required to induce ADCC by NK cells obtained from both types of donors. Thus, rituximab induced ADCC by FcgRIIIa 158V/V and 158F/F expressing NK cells at EC50s of 12 ng/ml [95% CI 11-13 ng/ml] and 31 ng/ml [95% CI 28-34 ng/ml], respectively. This 1.8 fold difference in ADCC potency between ofatumumab and rituximab was statistically significant (p<0.0001). Conclusions We assessed FcgRIIIa affinity and potency to induce ADCC by purified NK cells for ofatumumab and rituximab in a blinded study. Expected differences in affinity for the 158V and 158F allotypes of FcgRIIIa were observed for both ofatumumab and rituximab. These differences correlated with a stronger ADCC by FcgRIIIa 158V/V compared to 158F/F expressing NK cells. Significantly, ofatumumab was able to induce ADCC more potently than rituximab for both Fc receptor allotypes. Ofatumumab binds CD20 stably and at a distinct membrane-proximal epitope compared to rituximab. Our data suggest that these binding characteristics may positively impact ofatumumab's ability to direct killing of tumor cells via ADCC. Disclosures Craigen: GSK: Employment, Equity Ownership. Mackus:Genmab: Employment, Equity Ownership. Engleberts:Genmab: Employment, Equity Ownership. Miller:GSK: Employment, Equity Ownership. Speller:GSK: Employment, Equity Ownership. Chamberlain:GSK: Employment, Equity Ownership. Davis:GSK: Employment, Equity Ownership. McHugh:GSK: Employment, Equity Ownership. Bullmore:GSK: Employment, Equity Ownership. Cox:GSK: Employment, Equity Ownership. Wetten:GSK: Employment, Equity Ownership. Perdock:Genmab: Employment, Equity Ownership. Bakker:Genmab: Employment. van de Winkel:Genmab: Employment, Equity Ownership. Parren:Genmab: Employment.

The combination of chimeric CD20 monoclonal antibody (mAb) rituximab (R) with chemotherapy (CHOP) has significantly improved clinical outcome in patients with diffuse large B-cell lymphoma (DLBCL). However, the efficacy of R-CHOP is variable and DLBCL remains fatal in 30–40% of the patients. We recently developed the novel fully human CD20 mAb, ofatumumab (2F2) that is currently in clinical development for CLL and follicular lymphoma (phase III) and rheumatoid arthritis (phase II). In this study, we compared ofatumumab with rituximab for their ability to induce complement dependent cytotoxicity (CDC) of isolated lymphoma cells of chemotherapy-refractory DLBCL patients. Human type II CD20 mAb 11B8 does not induce CDC and was used as a control. Ofatumumab and rituximab induced CDC of all DLBCL samples tested, including the DLBCL cell lines SUDHL4, SUDHL5 and HT and lymphoma cells derived from ten chemotherapy-refractory DLBCL patients. Ofatumumab was significantly more effective in inducing CDC in nine out of ten DLBCL tumor samples when compared to rituximab (p=0.001, Two-way-Anova). The lethal doses (LD50) for ofatumumab (0.1 ± 2.8 μg/ml) was significantly lower when compared to the LD50 for rituximab (6.4 ± 4.9 μg/ml, p=0.04, Mann-Whitney test). The control CD20 mAb 11B8 showed no CDC induction of DLBCL cells. Sensitivity of DLBCL patient cells to ofatumumab- and rituximab-induced CDC negatively correlated with expression of complement defense molecule CD59, but not with expression of CD46, CD55, as determined by immunohistochemistry or apoptosis inhibitors Bcl-2 and XIAP. Functional inhibition of CD55 and CD59 using blocking mAb demonstrated that ofatumumab-induced CDC of DLBCL tumor cells was less sensitive to expression of these complement defense molecules than rituximab-induced CDC. We conclude that chemotherapy-refractory DLBCL cases are sensitive to CD20 mAb induced CDC with ofatumumab being the most effective mAb, especially in patients expressing high levels of CD59. Based on our results ofatumumab should be considered as a valuable therapy option for chemotherapy-refractory DLBCL patients.