Th eJournal of Translating Immunology Immunology

Translational Medicine in Action: Anti-CD20 in Sean H. Lim and Ronald Levy The introduction of for B cell lymphoma in plasma cell clonal progeny produce only one Ab with a single the late 1990s inaugurated a new era of cancer therapy specificity, that is, a mAb. In a normal immune response, showcasing mAbs. mAbs are in principle an amalgam- when many B cells are stimulated by Ag, multiple B cell ation of two characteristics of a perfect anticancer drug. clones develop, leading to multiple plasma cells, and the re- First, rituximab is a therapy targeted to the tumor cell, sultant Abs in the serum have diverse specificity for the Ag but it carries fewer side effects than does . (polyclonal Abs). In 1971, Norman Klinman (4) developed Second, with its ability to directly engage the host im- an in vivo/in vitro B cell cloning method that could produce mune system, it could potentially elicit longer lasting limited quantities of Abs in tissue culture from single cells, the anticancer immunity, although this remains to be proven. first mAbs (Fig. 1). In 1975, Ko¨hler and Milstein (5) made This review highlights the fundamental scientific discov- the critical breakthrough when they immortalized B cells by eries that allowed the development of clinically successful fusing them together with myeloma cells, thereby allowing the anti-CD20 mAbs. Since the approval of rituximab, a con- production of unlimited quantities of mAbs. This was a mo- mentous step in the history of immunology and it led to their siderable amount of work has been undertaken by dif- receipt of the Nobel Prize. However, even Ko¨hler and Mil- ferent groups trying to understand the workings and stein may not have initially realized that their discovery would limitations of anti-CD20s. All of these efforts will lead to drugs that would change the way we treat cancer. At be critical in designing new mAbs to CD20 and other about the same time, Stevenson and Stevenson (6) discovered targets and, ultimately, of anticancer mAbs that will that each malignant B cell, being derived from an ante- improve on, or even replace, chemotherapy. The cedent mature B cell, expressed a unique Ig on its surface that Journal of Immunology, 2014, 193: 1519–1524. could serve as a tumor-specific target. Therefore, in B cell lymphoma, all of the patient’s malignant cells can be specif- ymphoma is predominantly a cancer of B lymphocytes, ically recognized by their unique cell surface (idiotypic) Ig and nearly 70,000 new cases are diagnosed in the United structure. Soon thereafter we made mAbs against these by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. L States each year (1). Depending on the subtype of B tumor-derived idiotypes (7). When we administered these cell lymphoma, the disease can pursue an indolent course custom-made mAbs to patients with B cell lymphoma, most spanning years, such as in (FL), or it can responded to treatment and had durable remissions of their present aggressively during weeks or months, as in diffuse tumors (8–10). Despite its therapeutic efficacy, the need to large B cell lymphoma. Although it is acceptable to adopt produce a different Ab for each patient was impractical, al- a “watch and wait” approach in asymptomatic patients with though with newer technology it may one day be revisited. indolent disease, patients with more aggressive subtypes re- Earlier, in 1980, Nadler et al. (11) had infused a lymphoma quire immediate treatment. For more than three decades the patient with an mAb of undetermined specificity, but it was http://classic.jimmunol.org only therapeutic options available had been chemotherapy blocked by circulating cell-free substances and produced and/or radiotherapy. Response rates of 60–70% were achieved transient and clinically insignificant effects. In that same year but attempts to improve therapy by intensifying the number they discovered CD20, one of the first non-Ig B cell–specific of chemotherapeutic agents or their doses led only to more Ags (12). CD20 is highly expressed on normal and malignant side effects (2). Researchers had long recognized that novel B cells (12), and thus it could act as a target for mAbs in B cell were required, and immunologists were particularly lymphoma.

Downloaded from interested in the idea of a “magic bullet” (3), a drug that could Up to this point, all of the mAbs administered to patients specifically identify and kill cancer cells, thereby limiting toxic had been entirely of mouse origin. Surprisingly, patients with effects to normal, healthy cells. Several basic and ground- B cell lymphoma did not make Ab responses against the foreign breaking discoveries paved the way for the development of mouse proteins. However, for patients with lymphoma mAbs, the theoretical magic bullets. Abs are produced by (13, 14) and other types of malignancy, human anti-mouse terminally differentiated B cells known as plasma cells as part Abs against the murine components did arise, reducing of the healthy immune response. Each single B cell and its therapeutic efficacy and decreasing the survival of the thera-

Division of , Department of Medicine, Stanford University, Stanford, CA Abbreviations used in this article: ADCC, Ab-directed cellular cytotoxicity; CDC, 94305 complement-dependent cytotoxicity; CLL, chronic lymphocytic ; FL, follicular lymphoma; PML, progressive multifocal leukoencephalopathy. Address correspondence and reprint requests to Dr. Ronald Levy, Stanford University Medical Center, Center for Clinical Sciences Research, 269 Campus Drive, Stanford, Ó CA 94305. E-mail address: [email protected] Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1490027 1520 TRANSLATING IMMUNOLOGY: HISTORY OF CD20 BLOCKADE IN LYMPHOMA

FIGURE 1. Summary of the major events in mAb technology, together with mAb therapy developments that have led to chemoimmunotherapy as the standard of care in B cell lymphoma today. Blue boxes denote general advances in mAb technology; purple boxes, the discovery and progress of anti-idiotype mAb therapy; green boxes, rituximab-specific advances; and the red box denotes a description of next generation anti-CD20 mAbs.

peutic Ab (15). Surprisingly, no serious side effects from B cell lymphoma, rituximab was added to standard CHOP immune complexes of human anti-mouse Ab have been chemotherapy and it improved every measure of clinical documented (16). Later, advances in recombinant DNA outcome, including overall survival of the patients (23–26). In technology allowed the creation of chimeric mAbs, which low-grade lymphoma, rituximab shows clinical benefit even carried murine and human portions in their variable and when used as a single agent. The Swiss Group for Clinical constant regions, respectively (17). These developments Cancer Research demonstrated that single-agent rituximab, allowed the generation of C2B8, the first chimeric anti-CD20 administered weekly for four doses, then twice monthly for mAb, also eventually known as rituximab (18). This Ab was a further four doses, in newly diagnosed or relapsed/refractory able to deplete B cells in cynomolgus monkeys with no effects FL could establish long-term disease control (27). This has on other cell populations and with very little evidence of allowed low-grade lymphoma patients to defer chemotherapy, toxicity. In the early 1990s, we reported the first phase I or autologous transplantation, treatments with considerable clinical trial of rituximab (19). The Ab was administered as morbidity and mortality risks. In low-grade , a single injection at various doses to 15 patients with relapsed when rituximab was combined with chemotherapy as initial low-grade B cell lymphoma. The mAb was well tolerated and treatment (28, 29) or with salvage therapy (30), improve- 6 of 15 cases showed clinically significant tumor responses at ments in responses and survival are also achieved. Further-

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. all the doses and with no dose-limiting toxicity. These more, the use of rituximab as a maintenance dose every 2 mo, promising results led to a phase II multicenter study where for up to 2 y, after consolidation chemotherapy was both well 37 patients with low-grade B cell lymphoma were treated tolerated and delayed disease progression in responders, with with four weekly infusions of rituximab at 375 mg/m2 (20), a as many as 60% of patients free of disease progression 6 y dose chosen according to the maximum that could be easily after treatment (31, 32). Longer follow-up of these patients manufactured at the time. Seventeen patients showed tumor will be required to know whether this strategy of maintenance response to single-agent rituximab. Although normal B cells rituximab will result in longer overall survival of the patients. were depleted in the blood, the serum Ig levels did not fall and Frequently, new therapies come forward to the clinic before the patients did not show evidence of immunodeficiency. they are fully understood. Despite the success of rituximab in http://classic.jimmunol.org Considering that this population of patients had relapsed after the clinic, the mechanistic intricacies of the mAb still remain multiple rounds of chemotherapy, it was extremely promis- a matter of discussion and are extensively reviewed elsewhere ing that any of them responded to a single mAb. After a larger (33, 34). We need to understand how rituximab works against confirmatory trial that showed a remarkably consistent re- lymphoma and the mechanisms of tumor resistance if we are sponse rate (21), rituximab became the first mAb to be ap- to be able to make more potent Abs and to improve on the proved for the treatment of cancer by the U.S. Food and present results. Preclinical and clinical evidence both suggest Downloaded from Drug Administration. Of note, this approval was granted even that after anti-CD20 mAb binds to its target, CD20 on though no formal phase III trial comparing rituximab to other B cells, the Ab’s constant Fc domain binds to the host im- lymphoma therapies had been conducted. In part, this was mune effector cells such as NK cells and macrophages. These because of the relative freedom of rituximab therapy from immune effector cells either phagocytose the target cells di- serious side effects, in marked contrast with chemotherapy rectly or they release cytotoxic molecules such as perforin and drugs. Since the approval of rituximab, we estimate that .4 granzyme to lyse the target cells. These processes are known as million people have been treated with rituximab worldwide Ab-directed cellular cytotoxicity (ADCC) and phagocytosis, for lymphoma and other diseases such as rheumatoid arthritis. respectively. In line with this hypothesis, individuals who bear Czuczman et al. (22) first reported that rituximab could be Fc receptors with higher affinity to the Fc domain of Igs safely and efficaciously administered with chemotherapy in (FcRs) also have better responses to rituximab (35–37). Al- patients with low-grade lymphoma. The lack of toxicity on ternatively, anti-CD20 mAbs might kill by activating the host hematopoietic cells has also allowed rituximab to be safely complement pathway or directly inducing death signals in combined with chemotherapy, even in elderly patients (23). the cell upon binding to CD20 (reviewed in Ref. 34). Earlier In groundbreaking phase III clinical trials in diffuse large work suggested that anti-CD20 mAbs were capable of in- The Journal of Immunology 1521

ducing cell signaling and a “classical” form of cell death called rate (56). It is also not known whether trogocytosis would apoptosis and hence sensitization to cytotoxic chemotherapy. similarly apply to solid B cell malignancies where the tumor However, most of these earlier studies were performed in cell cells are less accessible to the peripheral vasculature than are lines and not primary tumor cells, or the mAb required ar- circulating CLL cells. tificial cross-linking (38-40). Recently published data show Another possible mechanism of rituximab resistance is the that cell death induced by anti-CD20 mAbs is in fact non- loss of surface CD20 expression after rituximab therapy, which apoptotic and is dependent on the type of anti-CD20 used might be due to transcriptional downregulation or selection of 2 (41). Finally, there is some evidence that anti-CD20 mAbs aCD20 clone (57-59). A further means by which target cells induce T cell–mediated immunity (42, 43). Consistent with can evade anti-CD20–mediated killing is by internalization this latter idea is the observation that responses to rituximab (59, 60) of the Ag/Ab complex into the target cell, thereby in patients can be delayed by as long as several months after preventing it from recruiting immune effector cells. Normal administration (20), a period of time that would allow induc- and malignant B cells express the inhibitory Fc receptor tion of T cell immunity. Furthermore, lymphoma patients FcgRIIb, which binds to the Fc domain of rituximab. To- who respond to rituximab the first time can have more du- gether the CD20/rituximab/FcgRIIb complex is then inter- rable responses on retreatment (20), a curious phenomenon nalized into the target cell and degraded. Furthermore, unlike that might be accounted for by the presence of memory normal B cells, which express a consistent level of FcgRIIb on T cells. the cell surface, malignant B cells have a heterogeneous ex- From the work of Cragg, Glennie, and colleagues (44–47) pression of FcgRIIb, with the level of FcgRIIb on the B cells we know that not all anti-CD20 mAbs are the same. They can correlating with the extent of rituximab internalization. Thus, be classified into either type I or II, depending on their it is possible that FcgRIIb expression on malignant B cells in vitro activity in various assays. This distinction is based on might serve as a biomarker for response to rituximab. Alter- the ability of the mAb to redistribute CD20 in Triton X-100– natively, internalization might be overcome by employing insoluble lipid rafts, induce homotypic adhesion, elicit com- type II anti-CD20s, which seem to internalize far less than do plement-dependent cytotoxicity (CDC), and/or direct cell death, type II anti-CD20s, or by combination of rituximab with an as discussed earlier. Type I mAbs such as rituximab are more anti-FcgRIIb inhibitor (60). potent at translocating CD20 into lipid rafts and inducing Recently, new anti-CD20s have entered the clinic. Ofatu- CDC, whereas type II mAbs such as tositumomab and mumab (type I) and (type II) were both ap- obinutuzumab are more effective at inducing homotypic proved by the U.S. Food and Drug Administration in 2009 adhesion and direct cell death induction through an actin- and 2013, respectively, for the treatment of CLL. Unlike dependent, lysosome-mediated pathway (41, 48). chimeric rituximab, both Abs are humanized, meaning they Evidently, understanding why anti-CD20s fail is as im- contain only the minimum critical murine sequences, that is, portant as knowing how they work. Approximately 30% of the CDR grafted into a human framework, with the theoretical cases of B cell non- either do not respond benefit being to reduce immunogenicity of mouse sequences. by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. to rituximab or relapse after initially responding, and they Ofatumumab is a type I mAb and it recognizes a distinct eventually become refractory to rituximab (reviewed in Ref. epitope, and it has a slower dissociation rate from CD20 49). We found that the level of the complement defense compared with rituximab, thus displaying greater CDC ac- molecules CD46, CD55, and CD59 on lymphoma cells did tivity than rituximab (47). As a consequence, ofatumumab is not correlate, either negatively or positively, with responses to able to lyse human lymphoma cell lines more effectively than rituximab (50). A series of comprehensive studies from does rituximab (47, 61). In contrast, obinutuzumab is a type Taylor and colleagues (51–53) showed trogocytosis as a po- II mAb with a sugar residue removed from its Fc domain tential mechanism of rituximab resistance. Trogocytosis is a to enhance Fc receptor binding (62, 63). Similar to other type http://classic.jimmunol.org process whereby phagocytic cells selectively remove bound II anti-CD20s, obinutuzumab is also superior in inducing Ag/IgG complexes (CD20/rituximab, in this case) from the lysosomal-mediated cell death, without the need for mAb surface of target cells rather than phagocytose them (51, 52). cross-linking (48). The two new anti-CD20 mAbs have dif- Consequently, the target cells are no longer opsonized and are ferent effects on immune effector cells (64). Obinutuzumab able to escape detection by immune effector cells. This process was superior at enhancing NK cell activation and ADCC is thought to be mediated by localized deposition of Ag/IgG whereas ofatumumab was better at inducing Ab-directed Downloaded from deposits, such as may occur owing to translocation of ritux- cellular phagocytosis by macrophages. These differences may imab into lipid rafts, as well as concomitant complement in part be due to a modified Fc domain of obinutuzumab that deposition, which aids FcR binding (54). In a proof-of- increases its binding affinity to FcgRIIIa on NK cells, but has principle study to support the evidence for trogocytosis, 12 less effect on FcgRIIa, the predominant activatory FcgR chronic lymphocytic leukemia (CLL) patients were treated present on macrophages (65). with lower doses of rituximab (20 or 60 mg/m2 three times The hope is that the new anti-CD20 Abs will be more a week for 4 initial weeks, as compared with conventional 375 efficacious than rituximab, but the clinical data so far are mg/m2 weekly for 4 wk). The study demonstrated that even at mixed. One study of a head-to-head comparison between doses as low as 20 mg/m2, rituximab was able to effectively rituximab and obinutuzumab in relapsed indolent lymphoma clear as much as 75% of the circulating CLL cells, and less showed no difference in progression-free survival (66). This trogocytosis was seen in this group compared with the group phase II study compared obinutuzumab at a dose of 1000 mg given 60 mg/m2 (55). These results, however, are at odds with to rituximab at the standard approved dose of 375 mg/m2. another previous study in CLL, which showed a dose–re- These differing doses were argued to be comparable in serum sponse relationship between rituximab and overall response levels achieved because of differing pharmacokinetics. Com- 1522 TRANSLATING IMMUNOLOGY: HISTORY OF CD20 BLOCKADE IN LYMPHOMA

1 parable overall responses of 32 of 74 and 29 of 75 patients reduced CD4 T cells. As many as 52 cases of PML have were reported. In a recent head-to-head comparison in been described in patients with lymphoma or CLL, and it patients with CLL (67), obinutuzumab was superior to rit- carries a 90% mortality rate (71). However, it needs to be uximab when combined with the drug chlorambucil, but stressed that the absolute risk is of PML is very low, and there again the doses and even the schedules of the two Abs were is little doubt that the clinical benefit of rituximab outweighs not matched. In short, although in vitro and xenograft studies its risks. support the potential of the new anti-CD20 mAbs over rit- uximab, the present trials are unclear as to whether these new Conclusions mAbs are indeed an advance. The dosing and schedule dif- Anti-CD20 mAb therapy has inaugurated a new era of cancer ferences between the mAbs being compared make it difficult (Fig. 1). In 1957, Burnet and Thomas first to conclude whether any observed clinical benefits are due to suggested that the host immune system is capable of elimi- differences in their mechanisms of action. nating spontaneously occurring tumors (reviewed in Ref. 72), Many early phase, single-arm studies of rituximab and and scientists have attempted in a myriad of different ways nonchemotherapeutic agents exist. As yet, none of these has to reawaken the host to fight cancer cells. There now exists replaced current conventional frontline rituximab-CVP or a well-tolerated drug that has the ability to re-engage the host rituximab-CHOP chemotherapy in low-grade and high-grade immune system and can be administered en masse to patients lymphoma, respectively. Neither has any of these approaches with B cell malignancies. Rituximab has indeed laid the been shown to be superior to standard salvage therapies. One of foundation for mAb therapy, and it is no surprise that all the strategies for enhancing the therapeutic effect of anti-CD20 attention is now focused on mAb therapy to many other mAbs is to administer a second mAb that stimulates NK cells different targets. The lessons learned from rituximab are im- responsible for ADCC. Preclinical studies show impressive portant for the clinical use of these future mAbs. No one therapeutic synergy between rituximab and an agonistic Ab single drug or mAb is going to “cure cancer” but rather the against CD137, a target appearing on NK cells once they see an future lies in new, scientifically rational combinations of drugs. Ab-coated tumor cell (68). Clinical trials are now underway to Anticancer therapy is currently still dominated by chemother- test this concept in patients with lymphoma. apy. Zitvogel et al. (73) have demonstrated that chemothera- The initial use of rituximab drew concerns with regard to peutic agents are not merely cytotoxic but are also able to alter possible immunosuppressive activity on the host, being that it the immunogenicity of tumor cells, which may explain why is a B cell–depleting mAb, and B cells are a vital part of our rituximab is more effective in combination with chemotherapy. body’s defensive adaptive immune system. Surprisingly, to Perhaps as we learn to understand the requirements for effective date, rituximab and the new anti-CD20 mAbs are generally anticancer therapy, newer mAbs will be able to extend the well tolerated. The most common side effects encountered are current ability to treat cancer while minimizing unwanted side related to the first mAb infusion, which is related to cytokine effects. release and is manifested as and chills. Infusion-related by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. events tend to be proportional to the patient’s tumor load and can be ameliorated by premedication with corticosteroids, Disclosures slower infusion rates, and smaller initial mAb doses. The The authors have no financial conflicts of interest. other notable side effect is late-onset neutropenia, the mech- anism of which is poorly understood and tends to be transient References and of significant consequence to the patient (reviewed in Ref. 1. American Cancer Society. 2013. Cancer Facts & Figures 2013. American Cancer 69). The risk of infections attributable to anti-CD20 therapy Society, Atlanta, GA. per se is more difficult to assess. Lymphoma in itself causes 2. Fisher, R. I., E. R. Gaynor, S. Dahlberg, M. M. Oken, T. M. Grogan, E. M. Mize,

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