Claude H. T. Chan

Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies

The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG(2)a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcgammaR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR-specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.

CD20 as a Target for Therapeutic Type I and II Monoclonal Antibodies

The last decade has seen the monoclonal antibody (mAb), rituximab, transform clinical management of many non-Hodgkin lymphomas and more recently provide new opportunities for controlling autoimmune conditions, such as rheumatoid arthritis. Although not yet fully determined, the explanation for this success appears to lie with the inherent properties of its target, CD20, which allow rituximab to recruit potent cytotoxic effectors with unusual efficiency. In this review we detail the properties of CD20 that make it such an effective therapeutic target and describe how different mAbs change the membrane distribution and internalization of CD20 and have distinct modes of cytotoxic activity.

Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.

Induction of cytosolic calcium flux by CD20 Is dependent upon B cell antigen receptor signaling

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and “hijack” the signaling potential of the BCR.

Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity

A major feature that distinguishes type I from type II anti-CD20 monoclonal antibodies (mAbs) and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 and the inhibitory Fcγ receptor, FcγRIIb, in a bipolar configuration. Here, we investigated whether mAbs directed at other B-cell surface receptors also engaged FcγRIIb and whether this interaction promoted internalization. Most mAbs engaged and activated FcγRIIb, with the strength of activation related to the level of mAb bound to the cell surface. However, engagement did not affect internalization of most mAb-ligated receptors, either in cell lines or primary chronic lymphocytic leukemia cells with the exception of CD19 and CD38. Furthermore, at high cell concentrations/density both cis and trans interactions between cell-surface bound mAb and FcγRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that FcγRIIb is engaged by many mAbs in both cis and trans configurations, triggering its activation, but that internalization via FcγRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.

Immunotherapy Targeting Inhibitory Fcγ Receptor IIB (CD32b) in the Mouse Is Limited by Monoclonal Antibody Consumption and Receptor Internalization

Genetic deficiency of the inhibitory Fc receptor, FcγRIIB (CD32b), has been shown to augment the activity of activatory FcγR and promote mAb immunotherapy. To investigate whether mAbs capable of blocking FcγRIIB have similar capacity, we recently generated a panel of specific anti-mouse FcγRIIB mAbs that do not cross-react with other FcRs, allowing us to study the potential of FcγRIIB as a therapeutic target. Previous work revealed a number of these mAbs capable of eliciting programmed cell death of targets, and in the present study we demonstrated their ability to promote target cell phagocytosis. However, in a variety of murine tumor models, anti-FcγRIIB mAbs demonstrated limited therapeutic activity despite optimized treatment regimens. Unexpectedly, we observed that the anti-FcγRIIB mAbs are rapidly and extensively consumed in vivo, both by the tumor and host cells, including B cells, leading to a precipitous loss from the circulation. Closer analysis revealed that the anti-FcγRIIB mAbs become extensively internalized from the cell surface within 24 h in vivo, likely explaining their suboptimal efficacy. Subsequent studies revealed that anti-FcγRIIB mAb immunotherapy was effective when used against FcγRIIB+ tumors in FcγRIIB−/− recipients, indicating that consumption of the mAb by nontumor cells is the primary limitation of these reagents. Importantly, similar rates of internalization were not seen on human target cells, at least in vitro. These studies further highlight the need to determine the propensity of mAb therapeutics to internalize target receptors and also identify potential key differences between human and mouse cells in this respect.

Activatory and inhibitory Fcγ receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain.

Background: Fcγ receptor (FcγR) IIb augments internalization of CD20 from the surface of B cells in response to rituximab treatment. Results: Activatory and inhibitory FcγR augment internalization, independent of the FcγR cytoplasmic domain. Conclusion: Active signaling is not required for FcγR-augmented internalization of CD20 in response to rituximab treatment. Significance: FcγR may play a structural role in augmenting CD20 internalization. Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.

Abstract LB-142: A trial of agonistic anti-CD40 antibody: Biological effects in a Cancer Research UK phase I study.

Introduction: This phase I study aimed to establish the biological effects and maximum tolerated dose (MTD) of the agonistic IgG1 chimeric anti-CD40 antibody, Chi Lob 7/4, in patients (pts) with a range of CD40 expressing solid tumors and diffuse large B-cell non-Hodgkin lymphoma (DLBL), resistant to conventional therapy. Potential mechanisms of action for agonistic anti-CD40 include direct cytotoxic effects on tumor cells, recruitment of immune effectors and conditioning of antigen-presenting cells Methodology: Chi Lob 7/4 was given by IV infusion weekly for 4 doses at a range from 0.5 to 240mg/dose. Validated ELISA9s were used to quantify Chi Lob 7/4 in serum and test for anti-chimeric MAb (HACA) responses. Pharmacodynamic assessments included quantitation of T-, NK-, and B-cell numbers and activation in blood by flow cytometry, a panel of cytokines in plasma by Luminex® technology and phenotypic assessment of human plasmacytoid and myeloid dendritic cells (DC) by a novel 6 colour flow cytometry panel Planned dose escalation was in cohorts of 3 pts until MTD or biological effect, defined as reduction of peripheral blood CD19+ B-cells to 10% or less of baseline. Results: Twenty-eight pts with CD40+ tumors were treated. 11 had mesothelioma, 3 melanoma, 2 oesophageal, 2 colorectal, 2 head and neck, 2 cervical, 2 pancreatic, 2 DLBL and one each lung and thymic carcinoma. One pt with mesothelioma was re-treated with 240mg at an interval of more than 3 years, having experienced prolonged disease stabilisation after initial treatment at the 1.6mg level. Doses of up to 160mg x 4 were well-tolerated, but two pts treated with 240mg experienced grade 3 elevation of hepatic transaminase levels after 1 dose, indicating dose-limiting toxicity. The dose was reduced to 200mg x 4, and 6 pts have been treated at this level, with 1 experiencing grade 3 elevation of liver enzymes after 2 doses but 5 completing 4 doses without difficulties. Infusion reactions occurred at 16mg doses, largely preventable by corticosteroid premedication. Chi Lob 7/4 levels were measurable at doses of 50mg and above. At 200mg, Cmax on day 1 was 47-60mcg/ml, with terminal half life > 7 days and levels > 30 mcg/ml detectable at 1 week. HACA responses were common at low doses (5-50mg) but absent at higher doses (160-240mg), coincident with dose-dependent partial depletion of peripheral blood CD19+ B-cells. Transient falls in blood NK-cells occurred after the first dose of Chi Lob 7/4 at 16mg or greater, with elevated MIP-1beta maximal at 3-6 hours. Dose-dependent elevations in IL-12 occurred above 160mg, rising throughout the period of treatment. Activation of NK cells and monocytes was detected by elevated CD54 expression at doses of 160mg or above, but there was no consistent change in T-cell markers. Response assessment at week 8 showed stable disease after 15 courses, and progression after 14. The median duration of disease stabilization was 6 months, maximum 37. The pt re-treated for progressive mesothelioma after 3 years remains with stable disease at 17 months after a single 240mg dose. Conclusion: This antibody elicits B-cell depletion, and there was evidence of consistent NK and macrophage activation. The dose-limiting toxicity was liver enzyme rise, with the MTD being 200mg/dose (range between 2.1 - 3.3 mg/kg based on patient body weight). Disease stabilization in some cases suggests that further testing is indicated in CD40-expressing malignancies. Citation Format: Peter W. Johnson, Ruth Challis, Ferdousi Chowdhury, Claude Chan, Anna Smith, Neil Steven, Ceri Edwards, Margaret Ashton-Key, Elisabeth Hodges, Alison Tutt, Christian Ottensmeier, Anthony Williams, Martin Glennie. A trial of agonistic anti-CD40 antibody: Biological effects in a Cancer Research UK phase I study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-142. doi:10.1158/1538-7445.AM2013-LB-142