Targeting CD20: Rituximab's Success Fuels Interest in New Agents

Mechanisms of Action of Anti-CD20 Agents

This figure depicts several ways in which monoclonal antibodies are thought to induce tumor-killing activity in B-cell malignancies.

ADCC indicates antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; FcγR, fragment crystallizable gamma receptor; IE, immunization effect; MAC, membrane attack complex; mAbs, monoclonal antibodies; NK, natural killer; PCD, programmed cell death.

In the early 1900s, German scientist Paul Ehrlich theorized that by selectively targeting disease-causing organisms, we might be able to specifically deliver a toxin to kill that organism. This “magic bullet” concept was realized to some extent for cancer therapy with the development of monoclonal antibodies.

CD20’s Role in the Immune System

The first anticancer antibody on the market was rituximab, which targets the membrane protein CD20 and, since its approval in the late 1990s, it has revolutionized the treatment of B-cell malignancies, with huge improvements in survival rates. However, despite its success, a large proportion of patients are rituximab-refractory, either failing to respond or eventually relapsing. This has prompted the development of newer CD20 agents with altered structures that are designed to improve upon rituximab’s performance.The T and B cells of the immune system are coated in thousands of cell-surface molecules. Among them is a group known as the cluster of differentiation (CD) proteins, which reflect the type and developmental stage of the cell to which they are attached, and which are recognized by anti-B- and T-cell antibodies.

CD20 as Anticancer Target

CD20 is a member of a group of at least 26 proteins in mice and humans— the membrane-spanning 4A family. CD20 is found on the surface of all B cells at almost all stages of their development. Like many CD proteins, its precise cellular function is still unclear; however, it plays an important role in B-cell development and maturation, and is suspected of acting as a calcium channel in the cell membrane.The idea of using monoclonal antibodies as cancer therapy originated in the late 1970s and offered the potential for specifically killing cancer cells through antibody binding to proteins attached to the cancer cell surface. CD20 was seen as an ideal target against which to design monoclonal antibodies for a number of reasons, among them:

• It is expressed by the vast majority of B-cell lymphomas.
• It is not present on any other cells besides B cells, thus limiting off-target effects.
• The first stage of B-cell development does not express CD20; therefore, any healthy B cells destroyed by CD20-targeted therapy can be readily replenished.

The Rituximab Revolution

Anti-CD20 monoclonal antibody therapy has proven to be very effective, particularly when used in combination with the chemotherapeutic regimen CHOP (cyclophosphamide, hydroxydaunorubicin [doxorubicin], Oncovin [vincristine sulfate], and prednisone).In 1997, the chimeric antibody rituximab (Rituxan) became the first CD20-targeted agent to be approved by the FDA for the treatment of B-cell malignancies. It remains a staple in cancer therapy today and is approved for use as a single agent in patients with certain types of B-cell non-Hodgkin lymphoma (NHL), namely follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), and in patients with chronic lymphocytic leukemia (CLL). Approval was based on clinical trials in relapsed or refractory B-cell NHL in which an overall response rate of 48% and a median duration of response of 11.2 months were observed.

The real success story with rituximab, however, has been the synergistic activity it appears to have with several forms of chemotherapy, the mechanism of which remains uncertain. It has been possible to incorporate rituximab into these chemotherapy regimens with dramatic improvements in efficacy and without any significant additional toxicity. Response rates over 90% and median time to progression of 82 months have been observed with R-CHOP combination therapy.

Despite these remarkable responses and the substantial effects on patient survival, there are several major issues with rituximab treatment. When used as a single agent, close to half of patients fail to respond, and complete responses are seen in less than 10%. Most significantly, the majority of patients who do initially respond to rituximab treatment will eventually relapse. A number of mechanisms for the development of resistance or relapse have been proposed⎯primary among them is that tumor cells can lose CD20 from their surface following treatment, resulting in CD20-negative tumor cells that are resistant to rituximab.

Honing the Field With New Agents

The rituximab-refractory population has been the focus of further research efforts to better understand the precise mechanism of action of rituximab and the reasons for resistance or relapse, which in turn has led to the development of newer anti- CD20 antibodies with the aim of providing more effective therapy for these patients.Since rituximab’s approval, much has been learned about the mechanisms of action of anti-CD20 antibodies and the clinical responses observed with rituximab. It is now understood that these antibodies induce tumor killing in several different ways:

• Antibody-dependent cellular cytotoxicity (ADCC) This is the best-characterized mechanism of action and arguably the most important. The antibody binds to the CD20 antigen on the surface of the target cell and elicits an immune response through its fragment crystallizable (Fc) region, which is recognized by the Fc receptors on the surface of the effector cells of the immune system.
• Complement-dependent cytotoxicity (CDC) CDC is an immune response that involves a series of proteins known as the complement system. Some of these proteins, known as the membrane attack complex (MAC), insert themselves into the target cell membrane and destroy the integrity of the cell, causing cell death.
• Programmed cell death (PCD) This mechanism involves direct killing of the target cell through induction of apoptotic cell death signaling pathways.
• Immunization effect The clinical response to CD20 antibodies is often late-acting and prolonged, suggesting that they may also induce an adaptive antitumor immune response, a so-called immunization effect, leading to prolonged antitumor immunity.

In recent years, research has focused on generating new anti-CD20 antibodies that have the potential to improve upon the clinical performance of rituximab, particularly in rituximab-refractory patients, through alterations in their structure that aim to enhance activity in one of the above effector functions.

A second generation of antibodies was designed to be humanized or fully human, in order to reduce immunogenicity. Ofatumumab (Arzerra) has proven to be more effective at killing both rituximab-sensitive and rituximab-resistant tumor cells than rituximab. Clinical trials in patients with relapsed or refractory CLL or FL demonstrated response rates of 58% and 13%, respectively.

Third-generation CD20 antibodies are also undergoing clinical testing. In addition to being humanized, these agents have been engineered with a modified Fc region designed to improve therapeutic performance by promoting the interaction with Fc receptors on immune effector cells. This may prove of particular importance in patients with genetic polymorphisms in the FCGR3A gene that encodes the low-affinity Fc gamma receptor IIIa (also known as CD16a). Certain single nucleotide polymorphic variants of this gene have been associated with significantly reduced responses following rituximab treatment.

This newest generation of agents includes AME-133v and obinutuzumab (GA101). However, obinutuzumab differs from others in this group in an important way that highlights another key area of CD20 research: It is a type II CD20 monoclonal antibody.

CD20 antibodies can be divided into two groups, based on their ability to redistribute CD20 into lipid rafts in the cell membrane and the types of immune effector functions that they elicit. Type I, or rituximab-like, antibodies redistribute CD20 into lipid rafts, while type II, or tositumomab-like, antibodies do not. In addition, type I antibodies potently activate CDC, while type II activate PCD much more strongly.

More Novel Strategies

Type II antibodies are currently significantly underexplored and only one—tositumomab⎯ is approved as part of the Bexxar radioimmunotherapy regimen. They are likely to be an important avenue of research, however, as clinical studies indicate they are more effective at killing tumor cells than type I antibodies are.Other strategies that are being investigated include multivalent and bispecific antibodies, which target both CD20 and antigens present on other immune effector cells, such as the T-cell antigen CD3. Multivalent antibodies undergoing preclinical testing include the bispecific antibody FBTA05 (Bi20), which has shown greater levels of cytotoxicity and is in phase I/II trials; 11B8/2F2(ScFvHL)4-Fc, a bispecific, tetravalent anti-CD20 antibody that is derived from both a type I antibody (2F2) and a type II antibody (11B8) to combine optimal efficacy in eliciting CDC and PCD; and HexhA20, a hexavalent anti-CD20 antibody based on veltuzumab.

The use of cytokines to enhance rituximab activity in B-cell lymphoma is also under study. For example, the combination of interleukin (IL)-12 and rituximab is being evaluated in phase I trials in refractory, relapsed NHL and in phase II trials in relapsed, low-grade NHL, while the combination of rituximab and granulocyte colony-stimulating factor is undergoing phase I testing in FL and phase I/II trials in relapsed, low-grade lymphoma. Impressive response rates and duration of response have been reported thus far.

Finally, researchers are evaluating ways to specifically improve anti-CD20 antibody- associated CDC. For example, combining rituximab with the mini-antibodies MB-55 and MB-59 against CD55 and CD59 (two complement inhibitory proteins), respectively, has enhanced rituximab’s antitumor activity in preclinical studies.

The Anti-CD20 Landscape

FDA-Approved Naked Antibodies

Rituximab

(Rituxan) Genentech/ Biogen Idec

The original CD20-targeting agent, rituximab is a chimeric monoclonal antibody that the FDA has approved for the treatment of B-cell malignancies. As a single agent, it is approved for use in CD20-positive, relapsed/ refractory non-Hodgkin lymphoma (NHL). It is also approved with the standard chemotherapy regimen CHOP (cyclophosphamide/doxorubicin/ vincristine/prednisone) for patients with CD20-positive diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Clinical investigation of rituximab remains robust, with more than 80 open phase III studies in which the agent is either a comparator or used in new ways. For example, rituximab is being evaluated in combination with lenalidomide and whether it can be delivered via subcutaneous administration (versus intravenous) in NHL.

NCT01650701, NCT01476787, NCT01200758

Ofatumumab

(Arzerra) GlaxoSmithKline

A fully human anti-CD20 monoclonal antibody, ofatumumab was approved by the FDA in 2009 for the treatment of patients with chronic lymphocytic leukemia (CLL) resistant to both the anti-CD52 antibody alemtuzumab and the chemotherapy drug fludarabine. More than 50 clinical trials of ofatumumab are under way as a single agent and in combination with other agents in a variety of B-cell neoplasms. For example, it is undergoing phase III testing in patients with chronic lymphocytic leukemia (CLL), FL, and NHL.

NCT01678430, NCT01200589, NCT01077518

FDA-Approved Conjugated Antibodies

Ibritumomab tiuxetan

(Zevalin)

Spectrum Pharmaceuticals

Zevalin is an anti-CD20 monoclonal antibody (ibritumomab) coupled to the radioactive isotope yttrium-90 or indium-111 that is FDA-approved for the treatment of relapsed/refractory low-grade or transformed B-cell NHL and previously untreated FL in patients who respond to first-line chemotherapy. It is being evaluated in a phase III trial in DLBCL, in phase II studies in several combinations for FL and NHL, and for the treatment of multiple myeloma.

NCT01510184, NCT00662948, NCT01207765

I-131 Tositumomab

(Bexxar) GlaxoSmithKline

This agent consists of the mouse monoclonal antibody tositumomab linked to iodine I-131 radiolabeled targeting CD20. The FDA has approved the regimen for the treatment of relapsed or refractory CD20-positive follicular NHL. Tositumomab is a type II CD20 antibody and represents the only such FDA-approved type II agent. Several clinical trials are under way, including phase II trials in FL, mantle cell lymphoma, and multiple myeloma.

NCT01389076, NCT01484093, NCT00135200

In the Pipeline

Obinutuzumab

(GA101) Genentech/Biogen Idec

This fully humanized anti-CD20 monoclonal antibody is currently the only type II anti-CD20 antibody in clinical development. A phase I/II study of patients with relapsed/refractory CD20-positive NHL/CLL has been completed and response rates were greater than 50%. The agent is currently being investigated in combination with various chemotherapy regimens in several large phase III trials in DLBCL, CLL, and NHL. It is also being evaluated in combination with the CHOP or fludarabine/cyclophosphamide (FC) chemotherapeutic regimens (G-CHOP/G-FC) in patients with relapsed/ refractory CD20-positive FL, where the agent has achieved response rates greater than 90%.

NCT01659099, NCT01332968, NCT01059630, NCT01287741, NCT01010061, NCT00825149

Ocaratuzumab

(AME-133v)

Mentrik Biotech

In January 2012, the humanized, anti-CD20 monoclonal antibody AME-133v received the generic name ocaratuzumab. Ocaratuzumab has a modified fragment crystallizable (Fc) region designed to enhance binding affinity for CD20 and increase antibody-dependent cellular cytotoxicity (ADCC) function. Phase I/II trials in patients with follicular NHL who failed previous rituximab therapy demonstrated response rates of more than 30%, with a median progression-free survival of 91 weeks. In 2011, the FDA granted ocaratuzumab orphan drug status. NCT00354926

FBTA05

(Bi20)

Fresenius Biotech/ Technische Universität München

FBTA05 is a bispecific, trifunctional antibody that targets both CD20 and the T-cell surface antigen CD3. It also has a modified Fc region that binds Fc receptors on antigen-presenting cells (APCs). This trifunctionality allows it to bring three different immune effector cells (B cells, T cells, and APCs) into a complex, which is thought to mediate B-cell lymphoma cell killing even with very low CD20 expression levels. It is currently undergoing phase I/II testing in B-cell lymphomas. NCT01138579

Veltuzumab

(Bi20) (IMMU-106) Immunomedics, Inc

Veltuzumab is a humanized, type I anti-CD20 antibody, which binds more strongly to CD20 and has a greater effect on complement-dependent cytotoxicity (CDC) than rituximab. In a phase I/II trial in patients with relapsed/refractory NHL, response rates ranged from 44% to 83%, depending on the type of cancer. Phase I/II clinical testing in NHL and CLL continues. NCT00546793, NCT01101581

Jane de Lartigue, PhD, is a freelance medical writer and editor based in Davis, California.

Key Research

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