Francisco J. Hernandez-Ilizaliturri, MD

Associate Professor of Medicine and Immunology,

Immunology,Roswell Park Cancer Institute,

Monoclonal antibodies (mAbs) that target key &ldquo;restricted&rdquo; antigens in cancer cells have become an integral part of the management of multiple hematologic and solid tumor malignancies. The use of mAbs (ie, rituximab or trastuzumab) in the treatment of patients with B-cell lymphoma or breast cancer has improved clinical outcomes and has changed the treatment paradigm of such conditions. CD20 antigen was identified as a suitable target for mAb development in lymphoid malignancies almost 3 decades ago. Rituximab, the first of three commercially available mAbs targeting CD20, has become a key component in the first-line or relapsed/refractory setting for various subtypes of B-cell lymphoma. While the clinical activity of rituximab is firmly established, scientific efforts had been focused on: (1) the optimization of its clinical use (ie, maintenance regimens); (2) improving its antitumor activity by combining it with novel chemotherapy agents, immunomodulatory agents, or smallmolecule inhibitors of key regulatory signaling pathways; or (3) defining the mechanisms by which rituximab kills lymphoma cells. Based on preclinical studies that characterized rituximab mechanisms of action, a new generation of humanized and more potent anti-CD20 mAbs has been generated. Obinutuzumab is a novel mAb against the CD20 antigen with promising clinical activity in patients with relapsed/refractory B-cell lymphoma (including rituximab-resistant lymphomas) and in those patients with previously untreated chronic lymphocytic leukemia. Obinutuzumab was re-engineered to elucidate better antibodydependent cellular cytotoxicity (ADCC) and a higher degree of apoptosis than rituximab, as demonstrated in laboratory models. In this article we discuss the preclinical and clinical development of this new, exciting agent.

The concept of using monoclonal antibodies (mAbs) to treat lymphoma was initially tested in the early 1980s when two independent groups of investigators reported the first cases of patients with lymphoma responding to a mouse anti-idiotype antibody.

Subsequent clinical studies were disappointing. Several factors contributed to such dismal outcomes: (1) suboptimal antigen selection (ie, modulation of the antibody-antigen complex or antigen shedding); (2) rapid clearance of antibody; and (3) development of xenograft immune reaction to the mAb (production of human antimouse antibodies by the host).

Cooperative efforts have produced a second and recently a third generation of mAbs with improved efficacy. Moreover, advances in molecular biotechnology and tumor immunology have led to the development of chimeric or humanized mAbs with increased biologic activity, longer half-lives, and less immunogenicity. Clinical trials have confirmed the improved antitumor activity of these, particularly rituximab and, recently, ofatumumab and obinutuzumab (Ob).

The structure and function of CD20 became an important area of scientific research after initial studies testing rituximab in relapsed/refractory Bcell lymphoma demonstrated meaningful clinical responses.

CD20 is a tetra-span transmembrane protein present in normal and malignant B cells. CD20 was one of the first B-cell specific differentiation antigens identified.

Laboratory studies suggest that CD20 is involved in B-cell activation and proliferation; on the other hand, CD20 knock-out mice exhibit a normal B-cell development.

The exact function of CD20 remains to be fully elucidated but it is believed to mediate intracellular calcium ion (Ca2+) influx and downstream cell signaling. However, given the restriction of its expression in B cells but not in bone marrow progenitor stem cells or in plasma cells, CD20 was identified as a suitable marker for targeting in B-cell malignancies.

Obinutuzumab (known also as GA101, Gazyva) is a glycolengineered, type 2 anti-CD20 humanized mAb that has exhibited higher activity than rituximab in B-cell lymphoma in preclinical studies.

Obinutuzumab was found to be active in relapsed/refractory follicular lymphoma, diffuse large B-cell lymphoma, and mantle cell lymphoma.

Results of a clinical trial comparing chlorambucil alone or in combination with rituximab or obinutuzumab in previously untreated elderly patients with chronic lymphocytic leukemia and multiple comorbid conditions demonstrated that obinutuzumab (at the dose schedule used for either anti-CD20 mAb) in combination with chlorambucil was more effective than either rituximab and chlorambucil or chlorambucil alone in terms of response rate and progression-free survival. Results of early clinical studies combining obinutuzumab and systemic polychemotherapy in relapsed/refractory B-cell lymphoma are encouraging.

Rituximab is an IgG chimeric mAb directed against CD20 antigen. As a result of initial clinical studies, rituximab became the first mAb to be approved by the Food and Drug Administration (FDA) to treat patients with CD20-positive B-cell lymphoma.

The early use of rituximab as a single agent or in combination with various systemic chemotherapy regimens (eg, cyclophosphamide, vincristine, and prednisone [CVP], cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP]) has resulted in improved response rates, duration of remission, progression-free survival (PFS), and overall survival (OS) in patients with follicular lymphoma (FL) and (with R-CHOP) in diffuse large B-cell lymphoma (DLBCL).

Similar observations have been seen in other lymphoid malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).

While most patients with B-cell lymphoma respond to rituximab or rituximab-based regimens, a significant number of them relapse after initial response or fail to exhibit a clinical response, stressing the need to further develop novel therapeutic strategies. The incorporation of rituximab into treatment algorithms for patients with B-cell lymphoma/ CLL has improved clinical outcomes, but has also changed the biology or clinical behavior of patients with primary refractory or relapsed disease. The need to develop novel salvage chemotherapy regimens after R-CHOP failures was further demonstrated by the results of the prospective multicenter, phase III Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) study. Investigators demonstrated that prior rituximab exposure in the frontline setting negatively affected the 4-year event-free survival (EFS).28 Moreover, the complete remission (CR) rate in R-CHOP pretreated patients with rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) or rituximab, or dexamethasone, cytarabine, and cisplatin (R-DHAP) was only 38%.

Scientific efforts need to focus not only on defining the pathways used by lymphoma cells to evade immunochemotherapy-based regimens, but also to develop novel therapeutic strategies to overcome or circumvent acquired resistance. Developing novel and more potent mAbs targeting CD20 is a promising therapeutic strategy in B-cell lymphoma in the post-rituximab era.

Several biologic effects have been postulated as rituximab&rsquo;s primary mechanism of antitumor activity, including: antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), antibody-dependent cellular phagocytosis (ADCP) by macrophages/monocytes, and/or induction of apoptosis/antiproliferation.

Preclinical studies have demonstrated that rituximab&rsquo;s primary mechanisms of action (MOA) in B-cell lymphoma are ADCC and CMC.

A better understanding of the MOA of rituximab can assist scientists/clinicians in the development of a new generation of anti-CD20 antibodies tested in clinical trials. In general, anti- CD20 antibodies can be classified as: (1) type 1 if upon binding to CD20 they show stabilization of CD20 into the lipid raft domains and high degree of complement activation (ie, rituximab, ofatumumab); or (2) type 2 when they show high homotypic adhesion and direct cell death (ie, Ob).

The new generation of anti-CD20 mAbs were designed to perform better than rituximab and have distinct antitumor properties. Alternative binding to CD20 at a different site (epitope) of the CD20 receptor or re-engineering of the constant region (Fc) of rituximab resulted in the development of two novel mAbs targeting CD20 (ofatumumab and Ob) that are entering the clinical arena. Our current contribution will focus on the preclinical and clinical development of Ob in B-cell lymphoid malignancies.

Preclinical studies had demonstrated that Ob has a higher activity than rituximab in terms of ADCC and induction of direct programmed cell death (

In B-cell lymphoma mouse models, Ob was found to be more potent than rituximab as a single agent or when combined with chemotherapeutic agents.

Two structural characteristics may explain the improved biologic activity of Ob over rituximab: (1) differences in the compartmentalization of CD20 across the cell membrane following antibody-receptor binding; or (2) differences in the structure of the Fc portion (

Structurally, Ob binds to the same epitope as rituximab. Interestingly, preclinical studies suggest that Ob binds CD20 in a completely different orientation than does rituximab, possibly affecting the spatial rearrangement of two CD20 molecules bound to a single molecule of Ob. Moreover, upon binding to CD20, Ob was found to induce redistribution of CD20 into areas of the cellular membrane associated with cell-cell contact interaction. Rituximab has the opposite effect (

It is unclear if these findings are associated with the improved antitumor activity of Ob observed in preclinical and clinical studies over rituximab.

On the other hand, the Fc region of Ob has an improved binding capacity to CD16 receptors present in neutrophils (CD16A) and in natural killer (NK) cells (CD16B), and therefore is capable of inducing better ADCC than does rituximab (

Mechanisms of action of obinutuzumab in B-cell malignancies. Structural differences FIGURE. (A) between obinutuzumab (type 2) and rituximab (type 1) leading different mechanisms of action (B) between these two biologically active monoclonal antibodies (mAbs).

Obinutuzumab is a type 2 glyco-lengineered mAb directed against CD20. While the variable region binds the same epitope of the CD20 antigen as rituximab, it binds to CD20 in a different orientation, leading to a different rearrangement of CD20 within the cell membrane and higher programmed cell death (apoptosis) than rituximab (type 2 vs type 1). In addition, the constant region (Fc) of the mAb is highly glycosylated, resulting in a higher affinity for the Fc gamma receptors (CD64 and CD16) present in polymorphonuclear (PMN) or natural killer (NK) cells, respectively. The glycosylated Fc region of obinutuzumab is capable of inducing a more effective antibody-dependent cellular cytotoxicity (ADCC). As a result, obinutuzumab&rsquo;s mechanisms of action are primarily ADCC and apoptosis, while rituximab is capable of inducing both ADCC and complement-mediated cytotoxicity with minimal apoptosis. NHL=non-Hodgkin lymphoma.

Taken together, the preclinical studies highlighted the superior antitumor activity of Ob when compared with rituximab in B-cell lymphoma. These studies paved the road for the clinical development of Ob and subsequent approval by the FDA for the treatment of relapsed/refractory CLL.

Obinutuzumab: Clinical Development (See summary in Table)

Obinutuzumab single-agent clinical studies The clinical activity of Ob had been evaluated in patients with CD20 lymphoproliferative disorders, primarily in patients with prior rituximab exposure.

evaluated the antitumor activity of Ob in 40 patients with relapsed/refractory non- Hodgkin lymphoma (NHL). Eligible patients were randomly assigned to receive Ob at a flat dosage of 400 mg on days 1 and 8 of each cycle (400 mg/400 mg) or at 1600 mg on days 1 and 8 of cycle 1 and then 800 mg on days 1 and 8 of subsequent cycles (1600 mg/800 mg).

Most of the enrolled patients had previously been treated with rituximab (38/40) and 22 of them were considered rituximab-refractory. While clinical antitumor activity was observed in the two dosage levels tested, higher overall response rates (ORRs) and CR rates were seen in the higherdose group (1600 mg/800 mg). The ORR was 55% for patients receiving Ob at the 1600 mg/800 mg dose-level and only 17% for the low-dose level (400 mg/400 mg). Complete responses were only seen in patients treated with the higher antibody dosages (9%). Six of 22 rituximab-resistant/refractory patients exhibited a clinical response to Ob, 5 of 10 in the 1600-mg/800-mg group and 1 of 12 in the 400-mg/400-mg group. The median PFS was 11.9 months in the 1600-mg/800-mg group (range, 1.8 to 33.9+ months) and 6.0 months in the 400-mg/400- mg group (range, 1.0 to 33.9+ months). The most common adverse events (AEs) were grade 1-2 infusion- related reactions (IRRs), observed in 73% of patients.

Obinutuzumab in combination with systemic chemotherapy for relapsed/refractory lymphoid malignancies

Parallel to this clinical trial, Morschhauser et al

reported the results of a second clinical study evaluating the antitumor activity of single-agent Ob in relapsed/refractory DLBCL and MCL.

In this trial, 40 patients with relapsed/refractory DLBCL (n = 25) and MCL (n = 15) were allocated in two dosage groups similar to the study design in the indolent lymphoma study (400-mg/400-mg and 1600-mg/800-mg dosage groups). In all, 21 patients received Ob at 400 mg on days 1 and 8 of each cycle, while 19 patients received Ob at 1600 mg on days 1 and 8 of the first cycle and then 800 mg on days 1 and 8 of each subsequent cycle. The ORR at the end of treatment was 28%, 32% for patients treated at the high-dosage level (1600-mg/800-mg group) and 24% for those patients receiving Ob at the lowerdosage level (400-mg/400-mg group). Activity was observed in both histologic subtypes. In addition, 20% of the patients with rituximab-refractory DLBCL or MCL exhibited a clinical response to Ob.

As reported in other Ob studies, grade 1-2 IRR were the most common AEs, while three patients had grade 3/4 IRRs. Grade 3/4 neutropenia was seen in only one patient.

The safety and antitumor activity of Ob in combination with systemic chemotherapy were evaluated in patients with previously untreated or relapsed/ refractory FL. Radford et al

reported the results of a phase IB study that explored the safety and efficacy of two dosage levels (1600 mg/800 mg [n = 28] or 400 mg/400 mg [n = 28]) of Ob in combination with either standard dosages of CHOP or fludarabine and cyclophosphamide (FC) in 56 patients with relapsed/ refractory FL. Patients were eligible if they had either relapsed or primary refractory FL, but a maximum of two prior chemotherapy-based regimens was required for entry into the study. There was no limit in the number of prior nonchemotherapy regimens (ie, mAbs or cytokines). Patients with relapsed/refractory FL requiring systemic therapy, based on the decision of the treating physician, were allocated to receive Ob 1600 mg on days 1 and 8 of cycle 1 and 800 mg on days 1 and 8 of subsequent cycles, or Ob 400 mg on days 1 and 8 of each cycle in combination with CHOP (Ob-CHOP) or FC (Ob-FC). Treatment cycles were administered every 3 weeks for 6-8 cycles for Ob-CHOP patients (n = 28) or every 4 weeks for 4-6 cycles for Ob-FC patients (n = 28). The primary endpoint of the study was safety, and secondary endpoints consisted of standard clinical outcomes (ie, ORR, CR rate), pharmacokinetics, peripheral B-cell depletion and recovery, and the impact of Fc&gamma; receptor polymorphisms in clinical outcomes.

While most patients had received rituximab in previous treatment, only 14 patients were rituximabrefractory as defined by the FDA (failed to respond or relapsed within 6 months from the last rituximab dose); four patients in the Ob-CHOP group and 10 patients in the Ob-FC group.

In general, Ob-CHOP and Ob-FC were well tolerated. Treatment discontinuation because of AEs occurred in five patients, two in the lower dosage of Ob and 3 in the 1600-mg/800- mg dose group. All of the patients who discontinued therapy received Ob-FC. On the other hand, all of the Ob-CHOP patients completed the planned therapy. The most common AE observed in patients treated with Ob-CHOP or with Ob-FC was neutropenia. Neutropenia was more commonly observed in patients receiving the higher dosage of Ob (1600 mg/800 mg) and in those patients receiving FC-containing chemoimmunotherapy. Grade 3-4 neutropenia was observed in 11 patients treated with Ob-CHOP and in 14 patients with Ob-FC. Most of those patients required growth factor support. IRR toxicities were the most common nonhematologic toxicities observed and occurred in 64% of the patients receiving Ob- CHOP and in 71% in the Ob-FC group.

Overall, AEs were more pronounced in patients receiving Ob-FC than Ob-CHOP.

Clinical activity was observed in most of the patients. At the end of treatment, 96% (27/28) of patients receiving Ob-CHOP (CR, 39% [11/28]) and 93% (26/28) receiving Ob-FC (CR, 50% [14 of 28]) achieved a clinical response. All of the patients with rituximab-refractory disease achieved at least a partial response (PR). The study demonstrated that Ob in combination with systemic chemotherapy is safe and effective in patients with FL and supports the design of subsequent randomized, phase III clinical trials in patients with relapsed/refractory FL.

Obinutuzumab was recently approved by the FDA for the treatment of previously untreated elderly patients with CLL.

The approval was based on the results of the CLL11 study, a large, randomized, phase III clinical trial comparing single-agent chlorambucil (Cl) to Cl in combination with rituximab (R-Cl) or Ob (Ob-Cl) in previously untreated CLL patients with multiple medical comorbidities. Treatmentna&iuml;ve CLL patients (N = 590) with a cumulative illness rating scale (CIRS) total score > 6 and/or an estimated creatinine clearance (CrCl) <70 mL/min were eligible. Patients were randomized (1:2:2 ratio) to receive Cl alone, Ob-Cl, or R-Cl. Chlorambucil was administered at 0.5 mg/kg orally on days 1 and 15 of a 28-day cycle for 6 cycles.

The dosages of Ob and rituximab were different in the combination groups. Patients in the Ob-Cl group received a higher cumulative dosage of an anti-CD20 mAb than did the R-Cl group. Obinutuzumab was dosed at 100 mg intravenously [IV] on day 1, 900 mg on day 2, and 1000 mg on days 8 and 15 of the first cycle of six 28-day cycles. For subsequent cycles (cycles 2-6), Ob was administered at 1000-mg fixed dosage. Rituximab was administered at 375 mg/m

on day 1 of the first R-Cl cycle and at 500 mg/m

TABLE. Summary of Obinutuzumab Clinical Studies in Lymphoid Malignancies.

relapsed/refractory indolent NHL (N = 40)

relapsed/refractory aggressive NHL (MCL and DLBCL) (N = 40)

Ob at dose levels and systemic chemotherapy (either CHOP or FC)

previously untreated CLL elderly patients (N = 781)

ORR: Cl (30.2%) vs Ob-Cl (75.5%) vs R-Cl (65.9%)

CRR: Cl (0%) vs Ob-Cl (22.2%) vs R-Cl (8.3%)

PFS Cl = 11.1 m vs Ob- Cl = 26.7 m (P < .001) or vs R-Cl = 16.3 m

CHOP = cyclophosphamide, doxorubicin, vincristine, and prednisone; Cl = chlorambucil; CLL = chronic lymphocytic leukemia; CRR = complete response rate; DLBCL = diffuse large B-cell lymphoma; FC = fludarabine and cyclophosphamide; FL = follicular lymphoma; MCL = mantle cell lymphoma; NHL = non-Hodgkin lymphoma; Ob = obinutuzumab; OS = overall survival; m = months. PFS = progression-free survival; and R = rituximab; ORR = overall response rate;

The primary endpoint was investigator-assessed PFS. Response rates, minimal residual disease (MRD), and OS were key secondary efficacy endpoints.

Results were analyzed in two stages; the first stage compared the clinical outcomes of patients in the control group to patients in the anti- CD20 mAbs groups (Cl vs Ob-Cl/R-Cl). The second stage compared the efficacy and safety of the headto- head comparison between Ob-Cl and R-Cl. The updated stage 1 analysis with longer observation time showed that Ob-Cl (median PFS = 26.7 months) or R-Cl (PFS = 16.3 months) had superior efficacy to chemotherapy with Cl alone (PFS=11.1 months). For patients with CLL, treatment with Ob-Cl resulted in improvement in the ORR and PFS but not in OS compared with R-Cl. The ORR and CR rates of patients treated with Ob-Cl were 78% and 21%, respectively. Treatment with R-Cl resulted in ORR and CR rates of only 65% and 7%, respectively. Moreover, the median PFS of patients treated with Ob-Cl (26.7 months) was longer than for those patients treated with R-Cl (15.2 months; P < .0001). The PFS benefit of Ob-Cl over R-Cl was observed in all preplanned subgroup analyses (including the cytogenetic subgroups 17p-, 11q-, 12+, 13q-).

The number of patients with MRD-negative blood samples at end of treatment was more than 10-fold higher with Ob-Cl than with R-Cl (29.4% vs 2.5%). Grade 3-4 IRRs with Ob-Cl occurred at first infusion only and were similar to those patients treated with R-Cl.

In summary, the results of this clinical trial further demonstrate that adding a mAb targeting CD20 (either rituximab or Ob) improves clinical outcomes, including for elderly patients with CLL and with multiple comorbid conditions. In this selected patient population, the use of Ob in combination with single-agent chemotherapy appears to improve the ORR, CR rates, and PFS over rituximab. It is unclear whether similar observations could be observed in younger patients with a more robust immune system and/or if equivalent dosages of rituximab and Ob are used.

In summary, Ob is a novel and potent mAb targeting CD20 with distinct structural and functional characteristics engineered to outperform rituximab in the clinical setting. Ongoing studies are aimed on further defining the better use of this exciting new biologic agent in previously untreated or rituximabrefractory B-cell lymphoid malignancies.

Nadler LM, Stashenko P, Hardy R, et al. Serotherapy of a patient with a monoclonal antibody directed against a human lymphomaassociated antigen.

Miller RA, Maloney DG, Warnke R, Levy R. Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody.

Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.

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Monoclonal antibodies (mAbs) that target key â€œrestrictedâ€ antigens in cancer cells have become an integral part of the management of multiple hematologic and solid tumor malignancies. 

Francisco J. Hernandez-Ilizaliturri, MD | Authors

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The Emerging Role of Obinutuzumab in the Treatment of B-Cell Lymphoid Malignancies