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.

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Increasing immune responses with immunostimulatory monoclonal antibodies (mAbs) directed to immune-receptor molecules is a new and exciting strategy in cancer therapy. This expanding class of agents functions on crucial receptors, either antagonizing those that suppress immune responses or activating others that amplify immune responses. Complications such as autoimmunity and systemic inflammation are problematic side effects associated with these agents. However, promising synergy has been observed in preclinical models using combinations of immunostimulatory antibodies and other immunotherapy strategies or conventional cancer therapies. Importantly, mAbs of this type have now entered clinical trials with encouraging initial results.

Recent success in cancer immunotherapy has reinvigorated the hypothesis that the immune system can control many if not most cancers, in some cases producing durable responses in a way not seen with many small molecule drugs. Agonistic CD40 monoclonal antibodies (mAb) offer a new therapeutic option which has the potential to generate anti-cancer immunity by various mechanisms. CD40 is a tumor necrosis factor receptor superfamily member expressed broadly on antigen-presenting cells (APC) such as dendritic cells, B cells, and monocytes as well as many non-immune cells and a range of tumors. Agonistic CD40 mAb have been shown to activate APC and promote anti-tumor T cell responses and to foster cytotoxic myeloid cells with the potential to control cancer in the absence of T-cell immunity. Thus, agonistic CD40 mAb are fundamentally different from mAb which block negative immune checkpoint such as anti-CTLA-4 or anti-PD-1. Initial clinical trials of agonistic CD40 mAb have shown highly promising results in the absence of disabling toxicity, both in single-agent studies and in combination with chemotherapy; however, numerous questions remain regarding dose, schedule, route of administration, and formulation. Recent findings regarding the role played by the IgG isotype and the Fc gamma receptor (FcγR) in mAb crosslinking, together with insights into mechanisms of action, particularly with regards to the role of myeloid cells, are predicted to help design next-generation CD40 agonistic reagents with greater efficacy. Here, we will review the preclinical and clinical data and discuss the major issues facing the field.

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CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40- lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.

Despite the clinical success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about its mechanism of action. Here we show that the ability of mAbs to translocate CD20 into low-density, detergentinsoluble membrane rafts appears to control how effectively they mediate complement lysis of lymphoma cells. In vitro studies using a panel of anti-B-cell mAbs revealed that the anti-CD20 mAbs, with one exception (B1), are unusually effective at recruiting human complement. Differences in complement recruitment could not be explained by the level of mAb binding or isotype but did correlate with the redistribution of CD20 in the cell membrane following mAb ligation. Membrane fractionation confirmed that B1, unlike 1F5 and rituximab, was unable to translocate CD20 into lipid rafts. In addition, we were able to drive B1 and a range of other anti-B-cell mAbs into a detergent-insoluble fraction of the cell by hyper-cross-linking with an F(ab) 2 anti-Ig Ab, a treatment that also conferred the ability to activate lytic complement. Thus, we have shown that an important mAb effector function appears to be controlled by movement of the target molecule into membrane rafts, either because a raft location favors complement activation by mAbs or because rafts are more sensitive to complement

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 heteroge- IntroductionThe anti-CD20 mAb rituximab has improved the overall survival of patients with follicular (FL) and diffuse large B-cell lymphoma (DLBCL). [1][2][3][4] However, in MCL, only modest responses are seen 5 and in CLL, fludarabine, cyclophosphamide and rituximab (FCR) therapy delivers improved responses but has yet to show a similar improvement in overall survival, 6 albeit the current follow-up is relatively short. Interestingly, those responses seen in CLL have often been achieved with high doses of rituximab, 6 suggesting that more mAb is needed to coat the targets or that it is consumed in some way. Even within rituximab-responsive lymphomas, a proportion of cases show resistance on first treatment with rituximab or eventually become resistant to rituximab-containing combination therapy (reviewed in Stolz et al 7 ). The molecular basis of this resistance and the observed sensitivity of different lymphoma subtypes is unclear (reviewed in Lim et al 8 ), but is highly relevant to improving outcomes.In addition to understanding target resistance, many groups are working to deliver anti-CD20 mAb reagents with improved affinity and more potent engagement of cytotoxic effectors. Anti-CD20 mAb can be defined as type I (eg, rituximab, ofatumumab) or type II (eg tositumumab, GA101), according to their ability to redistribute CD20 into lipid rafts in the plasma membrane and function in various effector assays. 9-11 It is still not clear what characteristics are required for the optimal reagent, but it is generally accepted that Fc:Fc ␥ receptor (Fc␥R) interactions are crucial to the efficacy of anti-CD20 mAb. [12][13][14][15] In particular, Fc␥RIIIa on myeloid effectors appears critical in controlling Ab potency and in keeping with this, lymphoma patients bearing the higher affinity 158V allele in Fc␥RIIIa respond better to rituximab compared with those with the low affinity 158F allotype, 16 leading many investigators to focus on augmenting the interaction of mAb with Fc␥RIIIa, for example via defucosylation. 17 Less attention has been given to the potential effects of the ITIM-containing inhibitory Fc␥R, Fc␥RIIb. Fc␥RIIb is a negative regulator of ITAM-containing receptors, such as the B-cell receptor (BCR) and the activatory Fc␥R. 18 Most hematopoietic cells coexpress inhibitory and activatory Fc␥R, and tumors are reported to be more sensitive to mAb immunotherapy in Fc␥RII Ϫ/Ϫ mice because of the removal of the inhibitory restraint of this receptor from myeloid effectors such as macro...