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Clinical Pharmacology

A Mechanistic Perspective of Monoclonal Antibodies in Cancer Therapy Beyond Target-Related Effects

^aDepartment of Otorhinolaryngology, Head and Neck Surgery, ^bMarlene & Stuart Greenbaum Cancer Center, and ^cDepartment of Pathology, Division of Immunogenetics, University of Maryland School of Medicine, Baltimore, Maryland, USA

Key Words. Cancer • Monoclonal antibodies • Antibody-dependent cellular cytotoxicity • Complement-dependent cytotoxicity

Correspondence: Scott E. Strome, M.D., F.A.C.S., Department of Otorhinolaryngology—Head and Neck Surgery, 16 South Eutaw Street, Suite 500, Baltimore, Maryland 21201-1619, USA. Telephone: 410-328-2378; Fax: 410-328-6192; e-mail: sstrome{at}smail.umaryland.edu

Received March 2, 2007; accepted for publication June 27, 2007.

Disclosure: S.E.S. owns stock in Gliknik, has acted as a consultant for Accuitive Medical Ventures, has performed contract work for GTC Biotherapeutics, and receives licensing revenue from IP agreements between Mayo Clinic and various third parties (as an inventor).

	Learning Objectives

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
After completing this course, the reader will be able to:

1. Describe the relationship between antibody structure and effector function, and identify strategies for modifying antibody structure to enhance these functions.
   
2. Explain how the efficacy of monoclonal antibodies in cancer therapy may occur via antibody- as well as target-related mechanisms.
   
3. Discuss how the ability of monoclonal antibodies to activate immune-mediated effector functions differs across antibody isotypes.
   

	ABSTRACT

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Several monoclonal antibodies are now in clinical use for cancer therapy, and many others are currently undergoing clinical evaluation. These agents offer unique specificity against key molecular targets on tumor cells or in the tumor microenvironment. The clinical efficacy of monoclonal antibodies is generally attributed to target-specific mechanisms resulting from neutralizing or inhibiting a growth factor or receptor that drives cell proliferation and tumor growth. Several targets, including CD20, human epidermal growth factor receptor 2, vascular endothelial growth factor, and epidermal growth factor receptor, have been validated in specific malignancies on the basis of monoclonal antibody efficacy. However, monoclonal antibodies also have the potential to activate immune-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. These functions result from interactions involving the Fc domain of the antibody, and, consequently, may vary by antibody, isotype, and Fc modification, such as changes in glycosylation. Accordingly, all monoclonal antibodies directed against a given target should not be considered equivalent in their ability to stimulate immune-mediated effector functions.

	INTRODUCTION

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Monoclonal antibodies (mAbs) became a therapeutic possibility for cancer with the development of hybridoma technology by Kohler and Milstein in 1975 [1]. This technology allowed the immortalization of antibody-producing cells derived from immunized mice and the subsequent selection of single-cell clones for the production of antibodies with high affinity and single specificity for an antigenic target. Early studies showed that murine mAbs directed against tumor antigens were effective in animal models, but translation of these findings into a clinical setting proved to be problematic [2]. The poor performance of mAbs in these early studies was attributed to short antibody half-life, immunogenicity of the murine protein in the human host, and depressed immune-mediated effector functions [3]. Importantly, it raised the question of whether mAbs directed against tumor antigens could elicit a sufficient immune response to promote clinically meaningful tumor regression.

Many of these early limitations were overcome by generating chimeric or humanized mAbs [2, 3]. A chimeric antibody contains the murine variable region (the part of the antibody that specifically recognizes its antigenic target) fused to constant domains of a human antibody backbone [4]. In comparison, a humanized antibody contains the murine sequence of only those sections of the variable domain that actually interact with the antigenic target. These so-called complementarity-determining regions (CDRs) are grafted onto a human antibody. Finally, fully human antibodies contain no murine sequences. Because most or all of the murine sequence has been replaced, the chimeric, humanized, and human antibodies are less immunogenic and may have longer half-lives because of a slower clearance [2].

Within the body, antibodies identify and label alien, potentially harmful particles, an initial step in the destruction of pathogens or abnormal cells. Subsequently, other components of the immune system attack and destroy the targets tagged by antibodies.

For therapeutic purposes, it was recognized that high-specificity binding by antibodies could neutralize membrane proteins regulating tumor growth. By blocking these growth factor receptors, antibodies could promote apoptosis or arrest growth of tumor cells merely by binding their target, thereby obviating the need to stimulate immune effector functions. These advances led to the development and approval of the first two mAbs for use in cancer therapy: rituximab (Rituxan®; Genentech, Inc., South San Francisco, CA) for non-Hodgkin's lymphoma in 1997 and trastuzumab (Herceptin®; Genentech, Inc., South San Francisco, CA) for breast cancer in 1998. Currently, at least six unconjugated mAbs are now approved by the U.S. Food and Drug Administration for use in cancer therapy (Table 1) [2, 5]. Several other approved mAbs are immunoconjugates, which are designed to deliver radioisotopes or cytotoxic agents specifically to tumor cells.

	ANTIBODY STRUCTURE AND EFFECTOR FUNCTION

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Antibody Structure and Isotypes
Antibodies are heterodimers consisting of two light chains and two heavy chains, in which each light chain is attached to a heavy chain by a disulfide bond, and the heavy chains are attached to each other by multiple disulfide bridges (Fig. 1) [6, 7]. The amino terminus of each light and heavy chain contains the variable region, which differs in amino acid sequence across antibodies. The unique specificity of antibodies depends on the amino acid sequence of the CDRs located within the variable region. Together, the CDRs on both the light and heavy chains form a unique structural conformation that represents the antigen-binding site of the antibody. The rest of the antibody molecule, known as the constant region, shows relatively few differences in amino acid sequence. On treatment with the enzyme papain, antibodies are degraded into two identical Fab fragments, each containing the antigen-binding site, and an Fc fragment without antigen-binding activity [7]. It is through the antigen-binding site that an antibody recognizes the antigenic epitope of its target, thus conveying its unique specificity. Whereas the antigen-binding site determines the antibody specificity, it is the Fc region that binds to effector cells or complement to trigger immune-mediated effector functions [6, 8].

View larger version (51K): [in this window] [in a new window]   Figure 1. Schematic of IgG antibody structure. Abbreviation: CDRs, complementarity-determining regions. From Sharkey RM, Goldenberg DM. Targeted therapy of cancer: New prospects for antibodies and immunoconjugates. CA Cancer J Clin 2006;56:226–243, with permission.

Immune-Mediated Effector Functions
Immune-mediated effector functions include two major mechanisms: ADCC and CDC. Both of them are mediated by the constant region of the immunoglobulin protein. The antibody Fc domain is, therefore, the portion that defines interactions with immune effector mechanisms.

ADCC
In ADCC, an IgG antibody first binds via its antigen-binding site to its target on tumor cells, and then the Fc portion is recognized by specific Fc receptors (FcR) on effector cells [3]. In humans, the FcR expressed on leukocytes include high-affinity FcRI (which binds to monomeric IgG and tends to be occupied by plasma IgG) and low-affinity FcRII and FcRIII (which bind IgG aggregates or immunocomplexes), each having several isoforms with differing cellular localization.

The intracellular structures of FcRIs and FcRIIIs contain activation domains that can stimulate immune cells via Src-family protein tyrosine kinases. Fc binding in the context of Fab ligation results in FcR crosslinking, thereby activating intracellular signaling and ultimately stimulating their effector functions. However, Fc binding to FcIIb, which is expressed by B cells, macrophages, and monocytes, induces an inhibitory signal that may serve to regulate effector functions. Natural killer (NK) cells are the principal effectors of ADCC; they express FcRIIc and FCRIIIa. The role of FcRIIc activation in NK cells is unclear, but activation of FcRIIIa induces ADCC and cytokine production [15]. ADCC is mediated by the release of cytotoxic granules, such as perforin, granulysin, and granzymes, whereas the release of cytokines and chemokines leads to inhibition of cell proliferation and angiogenesis. Macrophages also express FcRIIa and FcRIIIa, and can induce phagocytosis of antibody-coated tumor cells as well as promote ADCC through release of proteases, reactive oxygen species, and cytokines [3].

The ability of mAbs to stimulate ADCC depends on their isotype. IgG₁ and IgG₃ antibodies bind very well to FcRs, while IgG₄ and IgG₂ antibodies bind weakly (Table 2) [7]. Therefore, both IgG₁ and IgG₃ isotypes can provide a double-pronged therapeutic action: target-based and immune-based. IgG₃ antibodies, however, have a much shorter serum half-life (8 days versus 23 days for IgG₁s) probably due to different interactions between the two isotypes and the Fc neonatal receptor (FcnR) that regulates immunoglobulin homeostasis. This relatively short half-life makes the IgG₃ class suboptimal for therapeutic administration (except when short life poses an advantage; see immunoconjugates, below), and most mAbs currently available for cancer therapy are of the IgG₁ isotype (Table 1). These monoclonal IgG₁s allow for feasible administration and are most likely to promote ADCC, thus contributing an additional mechanism to their antitumor activity.

View this table: [in this window] [in a new window]   Table 2. Rank order of binding affinity of IgG isotypes for FcR subclasses [15]

CDC
CDC is another immune-mediated effector function that depends on antibody isotype. IgG₃ followed by IgG₁ are the most effective isotypes for stimulating the classic complement cascade: both isotypes bind to C1q leading to formation of C3b on the surface of antibody-coated tumor cells near the site of complement activation [7]. IgG₂ antibodies are less efficient in activating the complement cascade, whereas IgG₄ is unable to do so [4]. The presence of C3b controls formation of the C5–C9 membrane attack complex (MAC) that can insert into the membrane to lyse tumor cells. However, the enzymatic activity of C3b and consequently MAC formation are regulated by a series of membrane proteins that are overexpressed on many tumor cells [2]. These include CD35 (complement receptor type I), CD46 (membrane cofactor protein), and CD55 (decay accelerating protein), which inactivate the enzymatic activity of C3b, and CD59, which inhibits MAC formation; these markers have been demonstrated to inhibit tumor cell lysis by complement mediated by a therapeutic IgG₁ [16]. Therefore, a substantial contribution of CDC to the antitumor activity of mAbs may be unlikely, given the presence of these negative regulators in tumor cells [3]. Nevertheless, although C3b is inactivated to iC3b, its presence on the tumor cell surface may be recognized by effectors expressing receptors for C3 fragments, and in turn leads to synergistic interactions with FcR-mediated phagocytosis or ADCC [2, 17, 18].

Studying Immune Functions: Murine Models Versus Human Systems
Many of the studies that have shaped our understanding of immunologic functions have been carried out in murine models. It is worth considering, therefore, the substantial differences between the FcR system in humans and their murine counterparts. The FcR systems in mice and humans share a similar architecture, with a complex stimulatory/inhibitory receptor network, but the relative affinities of the stimulatory elements for each different isotype in mice do not match those in the human FcR family.

In mice, the FcRI is also a high-affinity ADCC-stimulatory receptor, but it binds preferentially the IgG₂ isotype (in particular, the subisotype IgG_2a) and seems to have a limited role in the functions of other murine IgGs [19, 20]. The activity of the murine FcRIII seems to be complementary to FcRI, binding the isotypes IgG₁ and IgG_2b more robustly than the IgG_2a [21–23]. Finally, a third stimulatory receptor, FcRIV, has high homology with the human FcRIII and moderate affinity for the IgG₂ isotype (a and b), but does not bind IgG₁ [24].

	MECHANISM AND ACTIVITY OF THERAPEUTIC MABS

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Target-Specific Mechanisms
Depending on the epitope against which an antibody is directed, antibody–antigen binding may neutralize circulating targets or cell surface receptors. Antibody binding to a receptor may prevent natural activation by ligands, or actually promote receptor activation. The epitope for antibody binding is very critical because some tumors may change surface proteins by post-translational modification, and consequently an antibody that recognizes a normal or unmodified antigen may no longer bind once the antigen has been modified. The currently available unconjugated mAbs are directed against molecular targets that are expressed on tumor cells or play an important role in the tumor microenvironment (Table 1).

Bevacizumab
Bevacizumab (Avastin®; Genentech, Inc., South San Francisco, CA) is a humanized IgG₁ mAb directed against vascular endothelial growth factor (VEGF). VEGF binds to VEGFR-1 and VEGFR-2 receptors located on vascular endothelial cells to stimulate excessive angiogenesis, thus allowing exponential tumor growth and providing a route for metastatic spread [25, 26]. By binding to VEGF, bevacizumab prevents VEGF from interacting with its receptors, and thus should inhibit new vessel growth. In preclinical models, bevacizumab blocked VEGF-induced cell proliferation, survival, and migration, reversed VEGF-induced vascular permeability, and normalized VEGF-induced changes in vessel architecture [27, 28]. These changes led to a reduction in interstitial pressure and increased blood flow, which may be important in improving the delivery of cytotoxic drugs used in combination with bevacizumab, and in reversing tumor hypoxia and its impact in mediating drug resistance. Interestingly, this effect may be short-lived, because tumor blood vessels eventually collapse after prolonged VEGF blockade, possibly leading to development of VEGF resistance [29].

Bevacizumab has demonstrated efficacy in metastatic colorectal cancer (mCRC), improving survival when added to a variety of cytotoxic platforms [30–32], and also in metastatic breast cancer [improving progression-free survival (10.97 mos. vs 6.11 mos.; HR = 0.498, p < 0.001) and possibly overall survival (HR = 0.674, p = 0.01) in combination with paclitaxel, but not capecitabine] [33, 34] and advanced non-small cell lung cancer (improving survival when added to carboplatin plus paclitaxel) [35].

Rituximab
Rituximab is a chimeric IgG₁ mAb directed against CD20, an antigen expressed on most B cells, including follicular B-cell lymphomas. In preclinical studies, binding of rituximab to CD20 has been associated with cell cycle regulation, altered expression of other cell surface molecules, and induction of apoptosis [36]. Although CD20 has been suggested to be a calcium channel involved in B-cell growth, its actual function is not entirely clear [37]. Therefore, it is not known whether any of the observed preclinical effects actually contribute to the clinical efficacy of rituximab [36].

Rituximab is effective in the treatment of lymphoma, providing progression-free and overall survival advantages when added to front-line cytotoxic chemotherapy for diffuse and other aggressive B-cell lymphomas [38, 39], as well as indolent forms of the disease (i.e., follicular) [40]. Rituximab is also an effective maintenance therapy in indolent lymphomas after response to initial therapy [41]. In addition, rituximab has produced promising results in the treatment of autoimmune diseases, including rheumatoid arthritis and certain lupus variants [42–44].

Ibritumomab tiuxetan and tositumomab also target CD20, but their efficacy may be related not only to target binding in itself, but also to their conjugated radioisotopes (see below).

	EPIDERMAL GROWTH FACTOR RECEPTOR–TARGETED ANTIBODIES: CETUXIMAB AND PANITUMUMAB

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
The other available mAbs target members of the erbB family of growth factor receptors: cetuximab (Erbitux^®, ImClone Systems Inc., Branchburg, NJ) and panitumumab (Vectibix^TM, Amgen Inc., Thousand Oaks, CA) are directed against the epidermal growth factor receptor (EGFR) and trastuzumab against human EGFR type 2 (HER-2). Following ligand binding, these receptors dimerize, leading to autophosphorylation, tyrosine kinase activation, and downstream signaling that ultimately leads to cell proliferation and tumor growth [45]. Cetuximab is a chimeric IgG₁ mAb; panitumumab is a human IgG₂ mAb. Both bind to the extracellular domain of EGFR, thereby acting as competitive antagonists of the natural ligands, EGF and transforming growth factor [45, 46]. As a result, these mAbs block EGFR-mediated signaling, leading to G₁ cell cycle arrest as a result of hypophosphorylation of the retinoblastoma protein [47]. In addition, these mAbs induce downregulation of EGFR expression on the cell surface [45].

Panitumumab is active as a single agent in the treatment of mCRC multirefractory to cytotoxics [48], but its efficacy as part of combination regimens is less clear [49]. Cetuximab is effective, in combination with cytotoxics and as single agent, in mCRC refractory to one or more therapies [50–52]. Cetuximab is also active in head and neck cancers, significantly prolonging survival when added to radiation therapy [53].

Trastuzumab
Trastuzumab is a humanized IgG₁ mAb that targets HER-2, which is overexpressed in some breast cancers. Binding of trastuzumab disrupts HER-2 signaling and blocks cell cycle progression in the G₁ phase, leading to inhibition of tumor growth [54]. The blockade of cell cycle progression by trastuzumab is correlated with the expression of p27^Kip1, an inhibitor of the cyclin E–CDK2 complex that controls progression through G₁ [55]. It remains unclear whether trastuzumab promotes HER-2 internalization or downregulation.

Trastuzumab is effective against metastatic breast cancer tumors overexpressing the HER-2 target, and its addition to standard chemotherapy results in higher response rates and longer progression-free survival (PFS) and overall survival times in this patient subpopulation [56–58]. The role of trastuzumab may extend to maintenance therapy throughout different cytotoxic regimens, but the benefit of that strategy is still to be determined [59].

These target-specific mechanisms—inhibition of VEGF by bevacizumab, binding to CD20 by rituximab, blocking EGFR by cetuximab and panitumumab, and blocking HER-2 by trastuzumab—are mediated by the antigen-binding site of the mAb and may contribute to some or all of the observed clinical efficacy of these agents. However, clinical efficacy may also be a result of, at least in some part, antibody-specific mechanisms mediated through the Fc domain of the mAb.

Antibody-Specific Mechanisms
Immune-mediated effector mechanisms, specifically ADCC and CDC, may contribute to the clinical efficacy of certain mAbs. Although preclinical evidence points to the potential importance of these mechanisms, there are only limited data to show that these mechanisms may indeed be important in the clinical setting.

ADCC
The strongest evidence supporting ADCC as a clinically meaningful mechanism of certain therapeutic mAbs is based on studies evaluating the impact of different allelic variations of FcRs (FcR polymorphisms) on clinical response (Fig. 2) [60, 61]. Polymorphisms have been identified in several FcR subclasses, including FcRIIIa and FcRIIa [37]. In FcRIIIa, a point mutation at nucleotide 559 results in substitution of valine by phenylalanine at amino acid 158. The FcRIIIa-158V/V protein has higher affinity for IgG₁, IgG₃, and IgG₄ and shows greater NK cell–mediated activity than the FcRIIIa-158F/F variant [62]. In FcRIIa, which is expressed on macrophages but not NK cells, a point mutation changes arginine to histidine at position 131 and results in higher IgG binding affinity, particularly for IgG₂ [15]. On the basis of the different binding affinities, patients harboring FcRIIIa-158V/V and FcRIIa-131H/H would be expected to mount a more vigorous ADCC antitumor response upon mAb treatment.

View larger version (19K): [in this window] [in a new window]   Figure 2. Response rates at 12 months with single-agent rituximab according to FcR genotype in patients with follicular lymphoma. Cartron and colleagues [60] evaluated 49 patients receiving first-line rituximab. Weng and Levy [61] evaluated 87 patients, including 72 patients who had received chemotherapy prior to rituximab. Abbreviations: F, phenylalanine; H, histidine; R, arginine; V, valine.

These findings were extended by Weng and Levy [61], who measured both FcRIIa and FcRIIIa polymorphisms in a cohort of 87 patients with follicular lymphoma, including 15 patients who received first-line rituximab and 72 patients previously treated with chemotherapy before rituximab. The cohort included 13 patients (15%) homozygous for FcRIIIa-158V/V and 34 patients (39%) homozygous for FcRIIIa-158F/F. Patients with the FcRIIIa-158V/V genotype had a significantly higher response rate than FcRIIIa-158F carriers when assessed during the first 3 months or at 6, 9, or 12 months (75% versus 26% at 12 months; p = .002). The 2-year PFS rate also favored the FcRIIIa-158V/V homozygotes over FcRIIIa-158F carriers (45% versus 12%; p = .023). When FcRIIa polymorphism was evaluated, 20 patients (23%) were homozygous for 131H/H and 24 patients (28%) for were homozygous for 131R/R. Patients with the FcRIIa-116H/H variant had a significantly higher 12-month response rate (55% versus 26%; p = .027) and 2-year PFS rate (37% versus 14%; p = .011) than FcRIIa-116R carriers. On logistic regression, both FcRIIIa-158V/V and FcRIIa-116H/H polymorphisms were independently associated with a higher response rate and longer PFS time on rituximab. Taken together, these studies support a role for ADCC in the clinical efficacy of rituximab; they are also suggestive of a proof of principle in which ADCC may be particularly important in the efficacy of other IgG₁ mAbs, including cetuximab, as this isotype is the most potent ADCC mediator.

Recently, Zhang and colleagues [63] explored whether FcRIIa and FcRIIIa polymorphisms would influence clinical response to single-agent cetuximab in 39 patients with mCRC who had failed previous irinotecan- and oxaliplatin-based therapy. The cohort included five patients (13%) with the FcRIIIa-158V/V genotype and nine patients (23%) with the FcRIIa-116H/H variant. Only two patients (5%) had partial responses to cetuximab—consistent with response rates reported for patients with advanced CRC. Patients with the FcRIIIa-158 F/F and F/V genotypes tended to have stable disease (57% and 71%, respectively), whereas those with the 158V/V genotype tended to have progressive disease (80%) on treatment with cetuximab (p = .082). Similarly, patients with FcRIIa-116H/H and H/R variants tended to have stable disease (78% and 71%, respectively), and those with 116R/R tended to have progressive disease (86%) (p = .082). Similar patterns were seen for PFS and overall survival, although the predictive value of the polymorphisms considered independently did not reach statistical significance. When the polymorphisms were considered together, however, patients harboring the combination of FcRIIIa-158V/V and FcRIIa-116R/R had a significantly shorter PFS interval than the remaining patients (1.1 versus 3.7 months; p = .004).

In principle, the impact of FcR polymorphisms on responsiveness to therapy supports a potential contribution of ADCC to antitumor efficacy for rituximab. Additional indirect evidence, albeit more limited, is beginning to indicate that ADCC may also be contributing to cetuximab activity. Taken together, however, these studies suggest that specific polymorphisms may influence outcomes differently with cetuximab in mCRC than with rituximab in follicular lymphoma: the presence of the FcRIIa-116H polymorphism seems to be indicative of a good response to therapy for both mAbs, while the FcRIIIa-158F variant is linked to a poor response to rituximab in patients with hematologic tumors, but it seems to be linked to a potent response to cetuximab in patients with mCRC. Despite the small sample size in these studies, it is important to recognize that not all mAbs are the same, even those of the same isotype. Potential differences may relate to the actual determinants of the antibodies or the exact mechanism(s) of cytotoxicity that they evoke.

Clearly, we are at an early stage in our understanding of whether ADCC contributes to the clinical efficacy of mAbs, and if so, to which ones. The interplay between host and tumor is, in all likelihood, the major determining factor in any immune response directed against tumors. Recent reports indicate that CRC tumors with a high density of infiltrating immune cells are less likely to disseminate; furthermore, the type, density, and location of immune cells that infiltrate into colorectal tumors are robust predictors of clinical outcomes, and their prognostic value is independent from, and superior to, staging based on histopathological criteria [64]. These findings point to the importance of immune responses in controlling tumor growth, on which therapeutic antibodies could capitalize. It is also important to consider that the intrinsic susceptibility of various tumor cells (e.g., colon cancer cells versus lymphoma cells) to immune-mediated effector functions may differ, and it may change as disease progresses. It is tempting to speculate that the benefit of ADCC may be greatest at earlier stages of disease when the tumor burden is smallest. Accordingly, it may be unrealistic to expect a major contribution to antitumor activity from ADCC in studies conducted with single-agent monoclonal therapy in advanced cancer, where immune function may be impaired by previous treatments or the nature of the disease itself [65]. Additional studies at earlier stages of disease are needed.

In theory, ADCC is less likely to be involved in the clinical response to an IgG₂ mAb such as panitumumab, than to IgG₁ mAbs. The FcRIIa-131H allele encodes the only receptor capable of efficiently interacting with IgG₂, and it is expressed on macrophages but not NK cells. Importantly, the FcRIIIa receptor on NK cells, regardless of the 158V/F polymorphism, binds poorly to IgG₂ [15]. Studies evaluating clinical response or outcome with panitumumab according to FcR polymorphism have not been reported to date.

CDC
Evidence supporting a role of CDC in the clinical efficacy of therapeutic antibodies, specifically rituximab, is based largely on preclinical models. Rituximab cured all wild-type mice injected with human CD20-transfected murine EL4 thymoma cells, but its protective effects were abolished in C1q-deficient mice lacking an intact complement pathway [17]. In contrast, depletion of NK cells or neutrophils did not influence the protective effects of rituximab, nor did testing in athymic nude mice. Similar findings were recently reported in a murine B-cell lymphoma model using human CD20-transfected 38C13 lymphoma cells [66]. Rituximab cured all animals with no evidence of lymphoma when assessed by immunohistochemistry and polymerase chain reaction analysis, whereas its protective effect was abolished after complement depletion with cobra venom factor. Again, depletion of NK cells or neutrophils, or removal of phagocytic macrophages, did not affect the protective action of rituximab. These models strongly suggest that the protective effects of rituximab depend on CDC, at least in the mouse (as discussed before, immunological differences between murine and human systems preclude immediate extrapolation of conclusions from one to the other). However, the role of CDC in the clinical efficacy of rituximab or other therapeutic mAbs remains unclear [18, 66]. Studies evaluating the importance of CDC with other therapeutic mAbs have not been reported.

As noted previously, CDC is regulated by a series of membrane proteins that promote inactivation of C3b or prevent MAC formation [2]. The role of these inhibitors in regulating the clinical activity of rituximab was explored in 29 patients with follicular lymphoma, most of whom had been treated with two or three courses of chemotherapy before receiving rituximab. Overall, eight patients had complete responses, 11 patients had partial responses, and the remaining 10 patients had no or minimal responses. The expression of the complement inhibitors CD46, CD55, and CD59, whether assessed alone or in various combinations, did not differ across the three response groups. Moreover, rituximab-induced CDC did not differ across the three groups when assessed in vitro [67]. Thus, the role of CDC in the clinical activity of rituximab, if any, is unclear based on available data.

	FUTURE TRENDS: OPTIMIZING THERAPEUTIC ACTIVITY

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Antibody Conjugates
Efforts to improve the cytotoxic actions of mAbs and consequently their therapeutic effectiveness have focused on conjugates with highly toxic substances, including radioisotopes and cytotoxic agents [6, 68, 69]. These conjugates can deliver a toxic load selectively to the tumor site while normal tissues are generally spared. In order to minimize toxicity, conjugates are usually engineered based on molecules with a short serum half-life (thus, the use of murine sequences, and IgG₃ or IgG₄ isotypes).

Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin®; Biogen Idec Inc., Cambridge, MA) and tositumomab (Bexxar®; GlaxoSmithKline, Research Triangle Park, NC) were approved in 2002 and 2003, respectively, in the U.S. for use in relapsed or refractory non-Hodgkin's lymphoma [70]. Both agents are murine mAbs that target the CD20 antigen on B-cell lymphoma cells (the same antigenic target as rituximab). Ibritumomab tiuxetan is an IgG₁ antibody that is conjugated to ⁹⁰Y, whereas tositumomab is an IgG₂ antibody containing ¹³¹I [71].

The conjugation of a cytotoxic agent to a mAb is illustrated by gemtuzumab ozogamicin (Mylotarg®; Wyeth Pharmaceuticals Inc., Philadelphia, PA) which was approved in the U.S. in 2000 for treatment of acute myelogenous leukemia (AML). This humanized IgG₄ mAb targets the CD33 antigen expressed in AML blast cells and contains a calicheamicin 1 derivative attached via a bifunctional linker [72]. Other conjugates of mAbs with cytotoxic toxins are under clinical evaluation [73]. For example, BL22 is a conjugate of an anti-CD22 mAb fragment and Pseudomonas exotoxin A, which has shown promising activity in chemoresistant hairy-cell leukemia [74]. Ricin has also been successfully conjugated with IgG₁ backbones targeted against CD22 (antibody RFB4–deglycosylated ricin A chain [dgA]) [75] and against CD19 (antibody HD37-dgA) [76]. Both of these molecules have clinical activity, and a combination is now under development for the treatment of pediatric and adult acute lymphoblastic leukemia [77].

Thus, mAb conjugates are a viable approach to killing tumor cells in hematological malignancies. However, this approach may be more problematic in solid tumors, where it may be difficult to deliver a sufficient amount of cytotoxic agent to achieve meaningful tumor regression [68]. Interestingly, the effect of conjugating a toxin or a radioisotope on the effector functions (ADCC or CDC) of an Ig moiety has not been consistently evaluated.

Enhancing Antibody Structure
Several strategies have been used to alter antibody structure in order to increase immune-mediated effector functions. The Fc region of IgG antibodies contains oligosaccharides that influence the orientation of the heavy chains, prevent interactions between adjacent domains, and allow exposure of key sugar residues on the antibody surface [78]. By maintaining the Fc domain in an open configuration, the presence of oligosaccharides—specifically, the N-linked oligosaccharide at asparigine-297 in the C_H2 domain of IgG₁—is important for binding to FcR as well as C1q [2, 4, 79]. Recombinant mAbs are commonly produced in Chinese hamster ovary cell lines, which generate oligosaccharides having a high fucose content [2, 80]. Through glycoengineering, mAbs with low fucose content can be produced. A defucosylated IgG₁ mAb against the chemokine receptor 4 exhibited greater binding to FcRIIIa, greater ADCC using human peripheral blood mononuclear cells or NK cells as effectors, greater phagocytic activity by monocytes and macrophages, and greater antitumor activity in murine models of T-cell leukemia and lymphoma than highly fucosylated IgG₁ mAbs [80, 81].

Modification of amino acids within the C_H2 domain of the Fc region is another strategy for enhancing immune-mediated effector functions. On high resolution mapping, several amino acids, all of which are located in the C_H2 domain near the hinge region, were identified as being important in IgG₁ binding to FcR [82, 83]. Several additional amino acids were also important in IgG₁ binding to FcRII and FcRIII. By changing these amino acids to alanine, variants with altered binding characteristics to FcRII and FcRIII were identified. The binding of IgG₁ to FcRIIIa, the main receptor mediating ADCC by NK cells, was 51% greater when simultaneous alanine mutations were made at Ser298, Glu333, and Lys334. Notably, this mutant exhibited greater ADCC mediated by NK cells, with cytotoxicity comparable to a 10-fold higher concentration of native IgG₁ [82]. It is hoped that by combining these antibody engineering strategies—reduced fucosylation and amino acid substitutions—it may be possible to generate mAbs with superior FcR binding characteristics, leading to more effective ADCC, which will ultimately translate into higher response rates and more durable responses, particularly in patients with solid tumors.

	CONCLUSION

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
mAbs represent an important advance in the treatment of certain hematologic malignancies and solid tumors. Unlike many small molecules, mAbs offer unique target specificity. The field has evolved rapidly in recent years, and now it is much easier to create mAbs against a variety of targets of potential relevance to tumor growth and survival. Targets, including CD20, HER-2, VEGF, and EGFR, have now been validated by the clinical efficacy of mAbs. In the future, the list of viable targets is likely to expand. Two areas that may merit more attention are membrane transporters and stromal function. Positron emission tomography scans detecting selective uptake of fluoroglucose by tumor cells are already being used diagnostically. By targeting specific transporters, mAbs may eliminate the ability of tumor cells to survive in a nutrient-challenged environment. The stroma is the interface between tumor and host, and accordingly, mAbs against stromal antigens may make it more resistant to the onslaught of tumor cells.

At first glance, the clinical efficacy of mAbs may be attributed to target-specific effects. By binding to their target, mAbs neutralize an important factor or receptor that drives cell proliferation and tumor growth. However, the therapeutic activity of mAbs may go beyond these target-related effects. Currently available mAbs are IgG antibodies, and consequently, they have the potential to activate immune-mediated effector functions, including ADCC and CDC. ADCC occurs when target-bound antibodies mobilize effector cells via interaction of their Fc domain with FcRs on the surface of immune cells. The interaction between the Ig Fc domain and FcRs on immune cells depends on the Fc domain (its sequence and glycosylation) and on the FcR structure (types and polymorphisms). Thus, the binding affinity of IgG for the FcgR mediating ADCC and other effector mechanisms varies by antibody isotype, and antibody-related effects may not be equal for all IgG isotypes or for all mAbs within a given isotype. The highest binding affinity for the various FcgR subclasses is found with IgG₁ and IgG₃, and therefore mAbs of these isotypes should be most likely to stimulate immune-mediated effector functions. Also, the intensity of ADCC is expected to fluctuate depending on the allelic FcR variants present in the host, and preliminary evidence points to an effect of those polymorphisms in clinical response to mAbs. Finally, by modifying antibody glycosylation patterns or amino acid sequence in the Fc domain, it may be possible to further enhance antibody-related effects, and hopefully, improve clinical efficacy of future mAbs.

	ACKNOWLEDGMENTS

Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 
Editorial assistance for the development of this manuscript was provided by Clinical Insights, Inc., with the financial support of Bristol-Myers Squibb and ImClone Systems, Inc.

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Top Learning Objectives Abstract Introduction Antibody Structure and Effector... Mechanism and Activity of... Epidermal Growth Factor Receptor... Future Trends: Optimizing... Conclusion Acknowledgments References

 

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