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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...

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR 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-DRspecific 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.Introduction mAbs are becoming increasingly utilized in the treatment of lymphoid disorders (1, 2). In particular, mAb directed to cell-surface antigens on malignant B cells has proven the most clinically effective, with the anti-CD20 mAb, rituximab, being the first to be approved by the US FDA for the treatment of cancer. Rituximab has substantially improved outcome for patients with many different types of non-Hodgkin lymphoma and has now been administered to over 1 million patients in the decade since its approval. Despite such success, treatment is not curative and there is intense preclinical and clinical investigation of many other engineered mAbs directed to both CD20 and a host of other cell-surface antigens (2).

Therapeutic antibodies have transformed cancer therapy, unlocking mechanisms of action by engaging the immune system. Unfortunately, cures rarely occur and patients display intrinsic or acquired resistance. Here, we demonstrate the therapeutic potential of targeting human (h) FcγRIIB (CD32B), a receptor implicated in immune cell desensitization and tumor cell resistance. FcγRIIB-blocking antibodies prevented internalization of the CD20-specific antibody rituximab, thereby maximizing cell surface accessibility and immune effector cell mediated antitumor activity. In hFcγRIIB-transgenic (Tg) mice, FcγRIIB-blocking antibodies effectively deleted target cells in combination with rituximab, and other therapeutic antibodies, from resistance-prone stromal compartments. Similar efficacy was seen in primary human tumor xenografts, including with cells from patients with relapsed/refractory disease. These data support the further development of hFcγRIIB antibodies for clinical assessment.

Key Points• Antigenic modulation significantly impacts natural killer cell and macrophage ability to mediate Fc g receptor-dependent killing.• hIgG1 mAbs are unable to elicit natural killer-mediated ADCC in the mouse, supporting ADCP as the dominant effector mechanism.Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. (Blood. 2015;125(12):1901-1909

Tumors routinely attract and co-opt macrophages to promote their growth, angiogenesis and metastasis. Macrophages are also the key effector cell for monoclonal antibody (mAb) therapies. Here we report that the tumor microenvironment creates an immunosuppressive signature on tumor-associated macrophages (TAM) which favors expression of inhibitory rather than activating Fcγ receptors (FcγR), thereby limiting the efficacy of mAb immunotherapy. We assessed a panel of TLR and STING agonists (a) for their ability to reprogram macrophages to a state optimal for mAb immunotherapy. Both STINGa and TLRa induced cytokine release, modulated FcγR expression and augmented mAb-mediated tumor cell phagocytosis in vitro. However, only STINGa reversed the suppressive FcγR profile in vivo, providing strong adjuvant effects to anti-CD20 mAb in murine models of lymphoma. Potent adjuvants like STINGa which can improve FcγR activatory:inhibitory (A:I) ratios on TAM are appealing candidates to reprogram TAM and curb tumor-mediated immunosuppression, thereby empowering mAb efficacy.

SummaryMonoclonal antibodies (mAbs) can destroy tumors by recruiting effectors such as myeloid cells, or targeting immunomodulatory receptors to promote cytotoxic T cell responses. Here, we examined the therapeutic potential of combining a direct tumor-targeting mAb, anti-CD20, with an extended panel of immunomodulatory mAbs. Only the anti-CD27/CD20 combination provided cures. This was apparent in multiple lymphoma models, including huCD27 transgenic mice using the anti-huCD27, varlilumab. Detailed mechanistic analysis using single-cell RNA sequencing demonstrated that anti-CD27 stimulated CD8+ T and natural killer cells to release myeloid chemo-attractants and interferon gamma, to elicit myeloid infiltration and macrophage activation. This study demonstrates the therapeutic advantage of using an immunomodulatory mAb to regulate lymphoid cells, which then recruit and activate myeloid cells for enhanced killing of mAb-opsonized tumors.

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.