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Mechanism of action and resistance to monoclonal antibody therapy
Mechanism of Action and Resistance to Monoclonal Antibody Therapy Neus Villamor, Emili Montserrat, and Dolors Colomer Monoclonal antibodies (MoAbs) are increasingly used in the treatment of patients with hematological malignancies and autoimmune diseases. The most commonly employed humanized and chimeric MoAbs are rituximab, alemtuzumab (Campath-1H, Ilex Pharmaceuticals, San Antonio, TX), and gemtuzumab-ozogamicin (Mylotarg, Wyeth-Ayerst Laboratories, St Davids, PA). The mechanism of action of these antibodies, and host and cellular factors influencing the response, are not completely known. Induction of apoptosis, antibody-dependent cell cytotoxicity (ADCC), and complement-mediated cell death (CDC) is the proposed mechanism of action of these antibodies. We review the current understanding of the mechanism of action of and resistance to these MoAbs. Semin Oncol 30:424-433. © 2003 Elsevier Inc. All rights reserved.
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REATMENT OF hematological malignancies has been largely based on chemotherapy and radiotherapy. Although improvement in response rates and survival has been obtained with these therapies over the years, a significant proportion of patients do not respond or relapse. Moreover, conventional cytotoxic therapy is often associated with significant morbidity. Recently, monoclonal antibodies (MoAbs) have emerged as important therapeutic agents in a number of hematological malignancies.1,2 MoAbs are proteins directed against specific antigens present on the surface of the cell. The main advantage of MoAb therapy compared to chemotherapy is that the former specifically targets cell surface antigens that are uniquely expressed on tumor cells or that are more highly expressed on cancer cells than on normal cells.3 IgG molecules
From the Unitat d’Hematopatologia and Servei d’Hematologia, Institut Clı´nic de Malalties Hematolo`giques i Oncolo`giques (ICMHO), Hospital Clı´nic. Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. Supported in part by Jose´ Carreras International Foundation Against Leukemia (EM/P-02), Roche Espan˜a, and by the Asociacio´n Espan˜ola Contra el Ca´ncer. Address reprint requests to Emili Montserrat, MD, Department of Hematology, Institute of Hematology and Oncology, Hospital Clı´nic, Villarroel 170, 08036, Barcelona, Spain. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3004-0013$30.00/0 doi:10.1016/S0093-7754(03)00261-6 424
are the most common antibodies employed in cancer therapy. They are composed of two identical light chains and two identical heavy chains, each consisting of variable and constant domains. The variable region contains the complementary-determining regions (CDRs), which are the major sites of interaction with antigens. The IgG molecule is divided into three functional domains: two antigen-binding (Fab) domains connected by a hinge domain to the effector Fc domain. The Fc domain interacts with seric complement components, immune effector cells, and receptors involved in maintaining IgG homeostasis.3 Three main classes of antibodies for therapy have been developed. The first consists of unconjugated MoAbs, where the antibody itself mediates cell killing. The other two classes are conjugated to a chemotherapeutic agent, immunotoxin, or radioactive particle. Conjugated MoAbs are designed to deliver a toxic agent to target cells, increasing its cytotoxic effect while minimizing side effects in nontargeted normal cells.3-5 An optimal target for MoAb therapy is an antigen highly expressed on the cellular surface membrane of the tumor cells with no presence as free protein in the plasma. It is necessary that, even when the antibody binds to the antigen, expression of the antigen remains at a high level on the target cells. On the other hand, the ability to induce immune responses with the production of human antimouse antibodies in the recipient, which block the therapeutic action of MoAbs, should be minimized. The development of humanized MoAbs, with the formation of chimeric proteins that have only a small antigen-binding mouse part and a large human constant Fc region, reduces their immunogenicity and prolongs their half-life. Since the introduction of rituximab, as the first targeted MoAb therapy for non-Hodgkin’s lymphomas (NHLs),6 MoAb therapy has become extensively used in patients with B-cell lymphoproliferative disorders.7,8 Rituximab (anti-CD20) and alemtuzumab (anti-CD52; Campath-1H, Ilex Pharmaceuticals, San Antonio, TX) have proven efficacy in different clinical trials.7,9-12 More reSeminars in Oncology, Vol 30, No 4 (August), 2003: pp 424-433
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cently, a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin (gemtuzumab-ozogamicin) has been approved for the treatment of CD33⫹ relapsed acute myeloid leukemia (AML).13,14 The purpose of this review is to discuss the main mechanisms of action of these three agents. ANTI-CD20 THERAPY
Rituximab is a chimeric humanized MoAb that contains a human IgG1 immunoglobulin constant region and a murine variable region specific for CD20. It binds to the surface antigen with the murine antibody part and stimulates the immune host mechanism through the human Fc portion.6 Rituximab has proven efficacy against a wide range of B-cell malignancies, including follicular lymphoma (FL), small lymphocytic lymphoma, marginal zone lymphoma, Waldenstro¨ m’s macroglobulinemia, mantle cell lymphoma, diffuse large cell lymphoma, and post-transplant lymphoproliferative disorders.7,8,15 CD20 is an integral transmembrane nonglycosylated hydrophobic phosphoprotein present on the surface of normal precursors and mature B cells; it remains expressed until terminal differentiation to plasma cells.16 CD20 is also present in the 90% of malignant B-cell NHLs and in about half of the cases with B-cell acute lymphoblastic leukemia; it is not expressed in neoplastic plasma cells of multiple myeloma.17 Importantly, CD20 expression is restricted to lymphoid B-cell lineage. The antigen is stable in the membrane of the B cell and does not shed, modulate, or internalize in response to antibody binding.6,18 The precise function of CD20 is still unknown, but it seems to play an important role in B-cell activation, differentiation, and growth.19-22 The CD20 cDNA predicts a tetraspan protein with intracellular NH2 and COOH termini and features of a membrane transporter or an ion channel.20 Cross-linking of CD20 results in modifications of B-cell growth and differentiation. However, the physiological CD20 ligands that would trigger similar responses have not been identified. The intracellular region of CD20 is rich in serine-threonine residues, contains multiple phosphorylation consensus sequences, and has been associated with the Src family kinases (Lyn, Fyn, and Lck), suggesting an implication of CD20 in transmembrane signaling pathways.23,24 It has been postulated that
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CD20 may be associated with lipid rafts.25-27 Lipid rafts are liquid-ordered membrane microdomains, enriched in sphingolipids and cholesterol, which are thought to function as platforms for signal transduction.28 In a recent study the association of CD20 to lipid rafts induced by rituximab produced a downregulation of the intracellular Lyn kinase.27 It has been postulated also that long-lasting perturbations of transmembrane signaling could contribute to progressive elimination of B cells.25,28 Mechanisms of Action of Rituximab Several mechanisms of action of rituximab have been proposed and analyzed in vitro, although the exact or predominant mechanism in vivo remains unknown. In vitro studies showed that rituximab is able to lyse CD20⫹ cells by inducing specific attack of tumor cells by immune cells through the Fc fixation (antibody dependent-cell mediated cytotoxicity [ADCC]) or by activation of the complement cascade (complement-dependent cytotoxicity [CDC]).29,30 In addition, rituximab induced apoptosis alone or in the presence of either goat antimouse IgG or Fc receptor-expressing cells.31,32 However, despite these in vitro data, whether the mechanism of action differs within compartments, and to what degree each of these mechanisms or other unrecognized mechanisms contributes to the destruction of tumor cells after treatment with rituximab, is unclear. ADCC and Rituximab ADCC is mediated by effector cells that express Fc receptors (FcR) such as natural killer (NK) and phagocytic cells. In vivo, FcR-expressing cells may infiltrate tumors and cross-link MoAb bound to the surface of tumor cells. ADCC and CDC are better mediated through the IgG1 constant region. The infusion in primates of an equivalent IgG4␥ version of rituximab is less effective in responses mediated by Fc and is unable to deplete B cells.33 Furthermore, there was a significant decrease in the antitumor effects of rituximab in Fc receptor– deficient mice.29 Three different classes of FcR for IgG (Fc␥R) have been described: Fc␥RI (CD64), Fc␥RII (CD32) and Fc␥RIII (CD16). Some FcR display a functional allelic polymorphism generating allotypes with different receptor affinities. Clinical responses of rituximab have been correlated with genetic polymorphisms identified on Fc␥RIIIa, an FcR present as a transmembrane mol-
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ecule on NK cells and monocytes. The polymorphism in Fc␥RIII, consisting of the presence in the amino acid position 158 of a valine (Val) or a phenylalanine (Phe), results in a different affinity for IgG1-Fc␥RIII binding. The Fc␥RIIIa-158 Val/ Val phenotype has greater affinity for human IgG1 than Fc␥RIIIa-158 Val/Phe or Phe/Phe.34 Among patients treated with rituximab alone for autoimmune diseases, those homozygous or heterozygous for the high-affinity allele achieve a greater depletion of B cells with the same plasma concentration of rituximab.35 In fact, 10-fold doses of rituximab are required in patients homozygous for the lowaffinity allele to obtain a similar reduction in B cells. In FL patients, the presence of homozygosity for Fc␥RIIIa-158Val is associated with a significantly higher objective response rate and a more frequent clearance of bcl-2 rearranged cells in both peripheral blood and bone marrow.36 Several groups have shown that incubation of tumor cells with rituximab and effector cells results in cellular lysis.30,37-39 Monocytes, NK cells, and neutrophils have been implicated in ADCC induced by rituximab,38 but the participation of different FcR in ADCC is not well understood. The recruitment of mononuclear cells seems to be mediated by CD16. Neutrophils normally express CD32 and CD16 and upon activation also express CD64. Tumor cell killing by neutrophils is more effectively triggered by CD64 and, as a result, is enhanced by granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colonystimulating factor (GM-CSF0.40,41 In addition, it has been described that phagocytosis of opsonized cells by macrophages is a process mediated by CD32; this process could be enhanced for cells less easily engulfed, such as chronic lymphocytic leukemia (CLL) cells, by the addition of GM-CSF.39 Finally, only NK cells are able to directly kill lymphoma cells in the presence of rituximab.38 In vitro assays of ADCC might not reflect what occurs during in vivo ADCC because ADCC experiments have been peformed in media depleted of complement, using effector:target cell ratios higher than expected in vivo30 and, normally, using allogeneic effector cells. Therefore, although these in vitro experiments show ADCC induced by rituximab, determination of its precise role in vivo requires additional investigation.
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CDC and Rituximab CDC is one of the mechanisms of action of rituximab. Through its human IgG1 Fc domain, rituximab can recruit serum proteins for CDC. The first component of the complement cascade, C1, is capable of binding the Fc portion of IgM and IgG molecules. C1 activation triggers the classical complement cascade, which leads to the formation of the membrane complex attack, which produces a direct lysis of the target cell. We and others have observed that CDC directly correlates with the expression of the CD20 antigen in malignant B cells, and the in vitro susceptibility to rituximab-mediated CDC depends primarily on CD20 protein expression (Fig 1).37,38,42 In fact, CDC is more rapidly and efficiently triggered by rituximab in cells with higher CD20 expression.37,42 It has been postulated that the lower rate of response to rituximab therapy in CLL patients compared with other lymphoproliferative disorders relates to the lower amounts of CD20 present on CLL cells. Therefore, the clinical differences of responses among patients could be related, at least in part, to CD20 expression. Deposition of complement proteins C337,43 and C937 is observed after in vitro treatment of B cells with rituximab in media containing complement, and colocalizes with rituximab.43 Whereas C3 deposition is observed independently of lytic responses and relates to CD20 expression, deposition of C9 and number of positive cells correlates with the degree of lysis.37 Moreover, the addition of anti-C3b(i) MoAb during in vitro experiments produces an increase in cell lysis.43 Furthermore, the administration of rituximab is followed by complement consumption.44 In a recent report analyzing gene expression in FL patients treated with rituximab, several complement proteins were among the genes differentially expressed between responders and nonresponders.45 The role of complement regulatory proteins in the modulation of rituximab efficacy has been addressed by different investigators.30,37,38,42,46 Several surface membrane proteins regulate the deposition of active complement proteins on cellular membrane to prevent cell lysis. Among these complement inhibitors, CD55 and CD59 seem to be the most important. No differences in the expression of CD59 molecules have been reported between normal B cells and malignant B cells from
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Fig 1. CDC is related to CD20 expression. (A) CD20 molecules per cell in responders (F) and nonresponders (E) in a series of patients with different B-cell lymphoproliferative disorders: CLL, FL, mantle cell lymphoma (MCL), and hairy cell leukemia (HCL). Response is mainly related to certain minimal amount of CD20 per cell. The gray bar highlights the zone where cells are heterogeneously sensible to rituximab. (B) Relationship between cell viability and CD20 expression after incubation with rituximab in the presence of complement. A direct correlation between CD20 molecules per cell and cytotoxicity was found.
different lymphoproliferative disorders,42 whereas low42 or high CD55 expression37 has been reported in CLL cells. Nevertheless, the in vitro susceptibility to rituximab-induced CDC could not be predicted by the levels of these proteins in CLL cells37,42 nor in vivo in FL47 and CLL patients.48 In contrast, using B-cell lines, a direct correlation between CDC and CD55 and CD59 has been reported by independent groups.38,49-51 Moreover, there is in vitro evidence that complement regulatory proteins play a role in the cytotoxic effect of rituximab (Fig 2). The blockage of CD55 and/or CD59 with specific antibodies sensitized cells to
Fig 2. CDC is increased after blockage with anti-CD55 and anti-CD59 monoclonal antibodies. Cells from five patients with CLL were incubated with rituximab and a complement source (Co) alone or with anti-CD55, anti-CD59 or both. Sensitization to rituximab was observed in all cases.
the cytotoxic effect of rituximab,37,42 indicating that these proteins, at least in vitro, may play an important role in rituximab’s action. Pretreatment with fludarabine52 or dexamethasone51 sensitizes cells to rituximab; it has been postulated for fludarabine that this is due to a decrease in the expression of CD55. All of these data suggest that analysis of CD55 and CD59 expression, in addition to that of CD20, might be useful to predict the response to rituximab in vivo. The ability of an anti-CD20 MoAb to recruit and activate complement is dependent not only on CD20 surface density or MoAb isotype, but also on the capacity of the antibody to translocate CD20 to lipid rafts.25 The mobility of CD20 molecules into lipid rafts would facilitate the engagement and activation of C1 and consequently the activation of complement cascade.25 CDC induced by rituximab results in cytoplasmic changes typical of apoptosis (ie, flow cytometry changes in forward side scatter (FSC) and side scatter (SSC) indicatives of cell shrinkage, exposure of phosphatidylserine residues, and decrease in the transmembrane mitochondrial potential). In contrast, no caspase activation and, consequently, no nuclear features of apoptosis are observed. Instead, a rapid and selective generation of reactive oxygen species (ROS) is detected after rituximab treatment. Finally, the use of ROS scavengers completely protects sensible cells to CDC induced by rituximab42 (Fig 3).
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Fig 3. Cellular modifications after incubation of malignant B cells with rituximab and complement (RⴙCo). (A) After 24 hours of in vitro treatment a loss of membrane integrity (propidium iodide–positive cells) an exposure of phosphatidylserine (annexin V–positive cells) is observed. (B) Loss of mitochondrial potential (DIOC6-negative cells) and generation of reactive oxygen species (ROS) (dihydroethidine-positive cells) in RⴙCo-treated cells. (C) Absence of caspase activation. No increase in cells expressing the active form of caspase 3 after RⴙCo treatment. (D) Incubation of cells with a ROS scavenger (N-acetyl-cysteine, NAC) protects cells from the RⴙCo effect. (E) Rapid generation of ROS after addition of a source of Co in cells pretreated for 10 minutes with rituximab.
Apoptosis and Rituximab Induction of apoptosis is another mechanism of action described for rituximab treatment. Apoptosis is observed after cross-linking,32,49 and some reports indicate that rituximab alone can induce apoptosis in B-cell lymphoma cell lines.31,38 The apoptotic process implicates the mitochondrial release of cytochrome c, loss of transmembrane mitochondrial potential, and activation of caspase-9 and the effector caspase-3.24,53 Although crosslinking of rituximab may lead to mitochondrial changes and caspase activation, these events are not necessary for relay of the apoptotic signal. It has been postulated that the apoptosis induced by anti-CD20 antibody in B-cell lines is dependent on increases in intracellular calcium, since apoptosis is inhibited using some extracellular calcium chelators.32 In patients with CLL, tumor cell clearance has been related to induction of apoptosis involving
the mithochondrial pathway and activation of caspases. After rituximab infusion, active forms of caspase-9, caspase-3, and poly-adenosine diphosphate ribose polymerase (PARP) are detected in peripheral blood of a proportion of these patients, followed by a decrease of lymphocyte counts. Furthermore, a significant downregulation of the antiapoptotic proteins XIAP and Mcl-1 is also observed.54 Recently, the Mcl-1/Bax ratio has been related to the clinical response in CLL patients.48 In some B-cell lines, downregulation of the antiapoptotic protein bcl-2 through interleukin-10 inhibition has been reported after rituximab incubation, rendering cells more sensitive to cytotoxic agents.55 These results suggest that the cytotoxic effect observed in vivo after rituximab treatment induces apoptosis, using a final pathway similar to chemotherapeutic agents.56 The differences in induction of apoptosis within anti-CD20 antibodies could be due to their differ-
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ent ability to segregate into rafts. Anti-B1, or tositumomab, is a anti-CD20 IgG2a that seems to bind an overlapping epitope of rituximab with a different affinity.49 In opposition to rituximab, tositumomab has shown significant antitumor activity and is able to induce apoptosis on Ramos cells in the absence of cross-linking.31,49,57-59 Cross-linking of tositumomab did not further increase cell cytotoxicity.49 These two antibodies differ also in their ability to induce CDC, which has been related with translocation to lipid rafts.25 Finally, dimerization of rituximab may also influence its ability to induce cell death. Thus, it has been observed that homodimeric rituximab dissociates more slowly than the monomeric form and only homodimers are able to induce apoptosis in cell lines.31 This could explain in part the differences observed in in vitro experiments. Mechanisms of Resistance to Rituximab In normal individuals, CD20 is not detected as a free protein in plasma. This situation is an ideal scenario for anti-CD20 MoAb therapy. Nevertheless, it has been recently shown that CLL patients may have significant amounts of circulating CD20. The presence of plasma CD20 is not due to active shedding or breakdown of cells and is supposed to circulate in large complexes or as fragments of cell membrane. The circulating CD20 levels vary among patients and seem to be associated with unfavorable clinical and biological parameters without correlation with lymphocyte counts. More importantly, circulating CD20 acts as a competitor for cellular CD20 in rituximab binding.60 Therefore, the presence of circulating CD20 might explain some treatment failures. A number of patients previously treated with rituximab who subsequently relapsed may display CD20⫺ tumors.61,62 The exact incidence of this phenomenon is unknown. In addition, a transient presence of malignant CD20⫺ B cells or modification in cell phenotype has been observed after rituximab treatment.63,64 This may explain some cases of resistance to re-treatment with rituximab. ANTI-CD52 THERAPY
CD52 antigen is a glycoprotein with an apparent molecular mass of 21 to 28 kd. It comprises a short peptide sequence of 12 amino acids and a large complex N-linked oligosaccharide that is attached to the membrane by a glycosylphosphati-
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dylinositol (GPI)-anchoring structure and is constitutively expressed in membrane lipid rafts. CD52 antigen is highly expressed on normal and neoplastic lymphocytes, monocytes, and macrophages, but not on erythrocytes or stem cells, and does not modulate.65 The antigen is also expressed on a large proportion of lymphoid cell malignancies. The function of CD52 remains unknown. Alemtuzumab (Campath-1H) is a recombinant DNA-derived humanized MoAb directed against the cell surface glycoprotein CD52. The alemtuzumab antibody is an IgG1 with human variable framework and constant regions, and CDR from a murine MoAb.66 Alemtuzumab treatment resulted in complete depletion of CD52⫹ cells, including B and T lymphocytes, monocytes, and NK cells from the peripheral blood. Alemtuzumab is increasingly used in the treatment of lymphoid malignancies, autoimmune diseases, and bone marrow transplantation, although it has a significant toxicity. Also, given its ability to deplete T cells, alemtuzumab has been used to prevent graft-versus-host disease following transplantation.67 Mechanism of Action of Alemtuzumab The mechanism of action of alemtuzumab has not been well established and this agent has been less extensively studied than rituximab. It has been proposed that CDC, ADCC, and apoptosis68-70 could mediate cell lysis. Cross-linking CD52 with the humanized MoAb alemtuzumab induces growth inhibition and apoptosis, the degree of which is related to the antigen density on the cell.68 In addition, the in vivo response to alemtuzumab has been correlated with CD52 expression on malignant cells.71 The mechanism of induction of proliferation arrest and apoptosis is not clear, but it could be mediated by tyrosine phosphorylation.68 The first dose-reaction observed after alemtuzumab was due to CD16 cross-linking of NK cells that release inflammatory cytokines including tumor necrosis factor-␣, interleukin-6, and interleukin-1.70 Despite the fact that they both carry the same human IgG1 Fc region, it has been suggested that rituximab and alemtuzumab differ significantly in their ability to activate human complement, with rituximab being a stronger activator than alemtuzumab.46 Complement activation is not completely necessary for depletion of CD52⫹ cells in vivo72 and cell lysis mediated by ADCC requires
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1,000-fold less antibody than CDC.73 This may be due to the secondary structure of each MoAb and its spatial relationship relative to the cell surface, which may affect its capacity to bind to C1 and activate the classic complement cascade.74 Mechanism of Resistance to Anti-CD52 Therapy After alemtuzumab treatment, the emergence of lymphocytes deficient in GPI-anchored proteins has been described.68,75-77 The degree of GPI-deficient cells and the time required to recover differs between B and T lymphocytes, with T cells being more frequently CD52⫺ than B cells.77,78 The phenotype of these CD52⫺ B and T cells closely resembles that of lymphocytes from paroxysmal nocturnal hemoglobinuria (PNH) patients, in which the first step of the GPI pathway is blocked due to mutations in the PIG-A gene. However, the molecular mechanism of the appearance of PNHlike clone after alemtuzumab is not well established. In CLL patients, a selection of cells carrying a PIG-A mutation present at low levels before treatment, and a decrease in mRNA PIG-A gene, has been described as a mechanism of emergence of PNH-like cells.75,76 Finally, some cases of CD52⫺ relapses in patients treated with alemtuzumab have been reported.79,80 ANTI-CD33 THERAPY
CD33, a myeloid differentiation antigen, is a member of the immunoglobulin superfamily and is a membrane-bound glycoprotein of 67 kd. CD33 is expressed by human monocytes, promyelocytes, myeloid blasts, and occasionally by acute lymphoblastic leukemias; it is absent from normal hematopoietic stem cells and nonhematopoietic tissues.81 Although it has been shown that the CD33 antigen is a sialic acid– binding receptor,82,83 its function on myeloid cells remains to be established. Gemtuzumab-ozogamicin (Mylotarg, WyethAyerst Laboratories, St Davids, PA) is a humanized IgG4 MoAb (hP67.6) against CD33 linked to a calicheamicin derivative (N-acetyl ␥1 calicheamicin).14 This molecule is a member of the enediyne family of antitumor antibiotics, which are more cytotoxic than other clinically used anticancer agents. Gemtuzumab-ozogamicin is rapidly internalized after binding to its target, followed by the release of the potent antitumor calicheamicin derivative. This compound-induced
double-stranded DNA breaks resulting in apoptosis.84 In this case, the mechanism of action may be different from that of rituximab and alemtuzumab. Gemtuzumab-ozogamicin induces DNA damage followed by cell cycle arrest in order to allow the cell to repair DNA or enter into apoptosis. It has been observed that within 3 to 6 hours after infusion, a near complete saturation of CD33 antigenic sites by is reached for AML blasts, monocytes, and granulocytes. After binding to CD33 antigen, gemtuzumab-ozogamicin is rapidly internalized and induces a dose-dependent apoptosis in myeloid cells in vitro.85 The response of AML cell lines to gemtuzumab-ozogamicin is heterogeneous. In some cell lines, gemtuzumab-ozogamicin induces an arrest in the G2 phase of the cell cycle, while other cell lines are blocked in G2 but rapidly undergo apoptosis and still others are resistant to gemtuzumab-ozogamicin. These different responses do not correlate with the levels of expression of CD33, and it has been proposed that the ATM/ATR-chk1/chk2 pathway is implicated in the cell cycle response to gemtuzumab-ozogamicin.86 No correlations between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including gemtuzumab-ozogamicin have been reported.87 Moreover, it has been described that residual marrow leukemia after gemtuzumab-ozogamicin treatment and failure to achieve complete or partial remission correlated highly with blast cells P-glycoprotein functions and with low in vitro drug-induced apoptosis.88 CONCLUSIONS
The use of MoAbs as targeted therapy for the treatment of hematologic malignancies has been increasing over the last few years. The mechanisms of action of these MoAbs are being increasingly better known. These include induction of apoptosis, ADCC, and CDC. The relative contribution of each mechanism and the role of genetic variability of the patients in the response to therapy remain to be determined. Advances in the understanding of the genetics of tumors and further investigation on the mechanisms of action of MoAbs will help in designing more specific and effective therapies.
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20. Einfeld DA, Brown JP, Valentine MA, et al: Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 7:711-717, 1988 21. Golay JT, Clark EA, Beverley PC: The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle. J Immunol 135:3795-3801, 1985 22. Tedder TF, Engel P: CD20: A regulator of cell-cycle progression of B lymphocytes. Immunol Today 15:450-454, 1994 23. Deans JP, Kalt L, Ledbetter JA, et al: Association of 75/80-kDa phosphoproteins and the tyrosine kinases Lyn, Fyn, and Lck with the B cell molecule CD20: Evidence against involvement of the cytoplasmic regions of CD20. J Biol Chem 270:22632-22638, 1995 24. Hofmeister JK, Cooney D, Coggeshall KM: Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. Blood Cells Mol Dis 26:133-143, 2000 25. Cragg MS, Morgan SM, Chan HT, et al: Complementmediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 101:1045-1052, 2003 26. Deans JP, Li H, Polyak MJ: CD20-mediated apoptosis: Signalling through lipid rafts. Immunology 107:176-182, 2002 27. Semac I, Palomba C, Kulangara K, et al: Anti-CD20 therapeutic antibody rituximab modifies the functional organization of rafts/microdomains of B lymphoma cells. Cancer Res 63:534-540, 2003 28. Brown DA, London E: Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275:17221-17224, 2000 29. Clynes RA, Towers TL, Presta LG, et al: Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 6:443-446, 2000 30. Harjunpaa A, Junnikkala S, Meri S: Rituximab (antiCD20) therapy of B-cell lymphomas: Direct complement killing is superior to cellular effector mechanisms. Scand J Immunol 51:634-641, 2000 31. Ghetie MA, Bright H, Vitetta ES: Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin. Blood 97:1392-1398, 2001 32. Shan D, Ledbetter JA, Press OW: Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 91:1644-1652, 1998 33. Anderson DR, Grillo-Lopez A, Varns C, et al: Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma. Biochem Soc Trans 25:705-708, 1997 34. Koene HR, Kleijer M, Algra J, et al: Fc gammaRIIIa158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood 90:1109-1114, 1997 35. Anolik JH, Campbell D, Felgar RE, et al: The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum 48:455-459, 2003 36. Cartron G, Dacheux L, Salles G, et al: Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood 99:754-758, 2002
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37. Golay J, Lazzari M, Facchinetti V, et al: CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: Further regulation by CD55 and CD59. Blood 98:3383-3389, 2001 38. Manches O, Lui G, Chaperot L, et al: In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas. Blood 101:949-954, 2003 39. Voso MT, Pantel G, Rutella S, et al: Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms. Haematologica 87:918-925, 2002 40. Stockmeyer B, Elsasser D, Dechant M, et al: Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies. J Immunol Methods 248:103-111, 2001 41. van der Kolk LE, de Haas M, Grillo-Lopez AJ, et al: Analysis of CD20-dependent cellular cytotoxicity by G-CSFstimulated neutrophils. Leukemia 16:693-699, 2002 42. Bellosillo B, Villamor N, Lopez-Guillermo A, et al: Complement-mediated cell death induced by rituximab in Bcell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species. Blood 98:2771-2777, 2001 43. Kennedy AD, Solga MD, Schuman TA, et al: An antiC3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20⫹ cells by rituximab. Blood 101:10711079, 2003 44. Winkler U, Jensen M, Manzke O, et al: Cytokine-release syndrome in patients with B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment with an antiCD20 monoclonal antibody (rituximab, IDEC-C2B8). Blood 94:2217-2224, 1999 45. Bohen SP, Troyanskaya OG, Alter O, et al: Variation in gene expression patterns in follicular lymphoma and the response to rituximab. Proc Natl Acad Sci USA 100:1926-1930, 2003 46. Golay J, Gramigna R, Facchinetti V, et al: Acquired immunodeficiency syndrome-associated lymphomas are efficiently lysed through complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity by rituximab. Br J Haematol 119:923-929, 2002 47. Weng WK, Levy R: Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular nonHodgkin lymphoma. Blood 98:1352-1357, 2001 48. Bannerji R, Kitada S, Flinn IW, et al: Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance. J Clin Oncol 21:1466-1471, 2003 49. Cardarelli PM, Quinn M, Buckman D, et al: Binding to CD20 by anti-B1 antibody or F(ab⬘)(2) is sufficient for induction of apoptosis in B-cell lines. Cancer Immunol Immunother 51:15-24, 2002 50. Treon SP, Mitsiades C, Mitsiades N, et al: Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies. J Immunother 24:263-271, 2001 51. Rose AL, Smith BE, Maloney DG: Glucocorticoids and rituximab in vitro: Synergistic direct antiproliferative and apoptotic effects. Blood 100:1765-1773, 2002
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52. Di Gaetano N, Xiao Y, Erba E, et al: Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone. Br J Haematol 114:800-809, 2001 53. van der Kolk LE, Evers LM, Omene C, et al: CD20induced B cell death can bypass mitochondria and caspase activation. Leukemia 16:1735-1744, 2002 54. Byrd JC, Kitada S, Flinn IW, et al: The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: Evidence of caspase activation and apoptosis induction. Blood 99:1038-1043, 2002 55. Alas S, Emmanouilides C, Bonavida B: Inhibition of interleukin 10 by rituximab results in down-regulation of bcl-2 and sensitization of B-cell non-Hodgkin’s lymphoma to apoptosis. Clin Cancer Res 7:709-723, 2001 56. Reed JC, Kitada S, Kim Y, et al: Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers. Semin Oncol 29:10-24, 2002 57. Golay J, Zaffaroni L, Vaccari T, et al: Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 95:3900-3908, 2000 58. Mathas S, Rickers A, Bommert K, et al: Anti-CD20 and B-cell receptor-mediated apoptosis: Evidence for shared intracellular signalling pathways. Cancer Res 60:7170-7176, 2000 59. Shan D, Ledbetter JA, Press OW: Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother 48:673-683, 2000 60. Manshouri T, Do KA, Wang X, et al: Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic significance. Blood 2002 61. Davis TA, Czerwinski DK, Levy R: Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression. Clin Cancer Res 5:611-615, 1999 62. Kennedy GA, Tey SK, Cobcroft R, et al: Incidence and nature of CD20-negative relapses following rituximab therapy in aggressive B-cell non-Hodgkin’s lymphoma: A retrospective review. Br J Haematol 119:412-416, 2002 63. Foran JM, Norton AJ, Micallef IN, et al: Loss of CD20 expression following treatment with rituximab (chimaeric monoclonal anti-CD20): A retrospective cohort analysis. Br J Haematol 114:881-883, 2001 64. Pickartz T, Ringel F, Wedde M, et al: Selection of B-cell chronic lymphocytic leukemia cell variants by therapy with anti-CD20 monoclonal antibody rituximab. Exp Hematol 29: 1410-1416, 2001 65. Xia MQ, Hale G, Lifely MR, et al: Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis. Biochem J 293:633-640, 1993 66. Hale G: The CD52 antigen and development of the CAMPATH antibodies. Cytotherapy 3:137-143, 2001 67. Dyer MJ: The role of CAMPATH-1 antibodies in the treatment of lymphoid malignancies. Semin Oncol 26:52-57, 1999 68. Rowan W, Tite J, Topley P, et al: Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells. Immunology 95:427-436, 1998 69. Xia MQ, Hale G, Waldmann H: Efficient complement-
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mediated lysis of cells containing the CAMPATH-1 (CDw52) antigen. Mol Immunol 30:1089-1096, 1993 70. Wing MG, Moreau T, Greenwood J, et al: Mechanism of first-dose cytokine-release syndrome by CAMPATH 1-H: involvement of CD16 (FcgammaRIII) and CD11a/CD18 (LFA-1) on NK cells. J Clin Invest 98:2819-2826, 1996 71. Ginaldi L, De Martinis M, Matutes E, et al: Levels of expression of CD52 in normal and leukemic B and T cells: Correlation with in vivo therapeutic responses to Campath1H. Leuk Res 22:185-191, 1998 72. Dyer MJ, Hale G, Hayhoe FG, et al: Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of antibody isotype. Blood 73:1431-1439, 1989 73. Riechmann L, Clark M, Waldmann H, et al: Reshaping human antibodies for therapy. Nature 332:323-327, 1988 74. Burton DR, Woof JM: Human antibody effector function. Adv Immunol 51:1-84, 1992 75. Fracchiolla NS, Barcellini W, Bianchi P, et al: Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy. Br J Haematol 112:969971, 2001 76. Rawstron AC, Rollinson SJ, Richards S, et al: The PNH phenotype cells that emerge in most patients after CAMPATH-1H therapy are present prior to treatment. Br J Haematol 107:148-153, 1999 77. Brett S, Baxter G, Cooper H, et al: Repopulation of blood lymphocyte sub-populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody, CAMPATH-1H. Immunology 88:13-19, 1996 78. Taylor VC, Sims M, Brett S, et al: Antibody selection against CD52 produces a paroxysmal nocturnal haemoglobinuria phenotype in human lymphocytes by a novel mechanism. Biochem J 322:919-925, 1997 79. Tuset E, Matutes E, Brito-Babapulle V, et al: Immunophenotype changes and loss of CD52 expression in two patients
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with relapsed T-cell prolymphocytic leukaemia. Leuk Lymphoma 42:1379-1383, 2001 80. Birhiray RE, Shaw G, Guldan S, et al: Phenotypic transformation of CD52(pos) to CD52(neg) leukemic T cells as a mechanism for resistance to CAMPATH-1H. Leukemia 16: 861-864, 2002 81. Peiper SC, Ashmun RA, Look AT: Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen. Blood 72:314-321, 1988 82. Taylor VC, Buckley CD, Douglas M, et al: The myeloidspecific sialic acid-binding receptor, CD33, associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2. J Biol Chem 274:11505-11512, 1999 83. Ulyanova T, Blasioli J, Woodford-Thomas TA, et al: The sialoadhesin CD33 is a myeloid-specific inhibitory receptor. Eur J Immunol 29:3440-3449, 1999 84. Nicolaou KC, Stabila P, Esmaeli-Azad B, et al: Cellspecific regulation of apoptosis by designed enediynes. Proc Natl Acad Sci USA 90:3142-3146, 1993 85. van DV V, te Marvelde JG, Hoogeveen PG, et al: Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells. Blood 97:3197-3204, 2001 86. Amico D, Barbui AM, Erba E, et al: Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin (Mylotarg) in vitro: Role of Chk1 and Chk2 phosphorylation and caspase 3. Blood 101:4589-4597, 2003 87. Jilani I, Estey E, Huh Y, et al: Differences in CD33 intensity between various myeloid neoplasms. Am J Clin Pathol 118:560-566, 2002 88. Linenberger ML, Hong T, Flowers D, et al: Multidrugresistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 98:988-994, 2001
T
REATMENT OF hematological malignancies has been largely based on chemotherapy and radiotherapy. Although improvement in response rates and survival has been obtained with these therapies over the years, a significant proportion of patients do not respond or relapse. Moreover, conventional cytotoxic therapy is often associated with significant morbidity. Recently, monoclonal antibodies (MoAbs) have emerged as important therapeutic agents in a number of hematological malignancies.1,2 MoAbs are proteins directed against specific antigens present on the surface of the cell. The main advantage of MoAb therapy compared to chemotherapy is that the former specifically targets cell surface antigens that are uniquely expressed on tumor cells or that are more highly expressed on cancer cells than on normal cells.3 IgG molecules
From the Unitat d’Hematopatologia and Servei d’Hematologia, Institut Clı´nic de Malalties Hematolo`giques i Oncolo`giques (ICMHO), Hospital Clı´nic. Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. Supported in part by Jose´ Carreras International Foundation Against Leukemia (EM/P-02), Roche Espan˜a, and by the Asociacio´n Espan˜ola Contra el Ca´ncer. Address reprint requests to Emili Montserrat, MD, Department of Hematology, Institute of Hematology and Oncology, Hospital Clı´nic, Villarroel 170, 08036, Barcelona, Spain. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3004-0013$30.00/0 doi:10.1016/S0093-7754(03)00261-6 424
are the most common antibodies employed in cancer therapy. They are composed of two identical light chains and two identical heavy chains, each consisting of variable and constant domains. The variable region contains the complementary-determining regions (CDRs), which are the major sites of interaction with antigens. The IgG molecule is divided into three functional domains: two antigen-binding (Fab) domains connected by a hinge domain to the effector Fc domain. The Fc domain interacts with seric complement components, immune effector cells, and receptors involved in maintaining IgG homeostasis.3 Three main classes of antibodies for therapy have been developed. The first consists of unconjugated MoAbs, where the antibody itself mediates cell killing. The other two classes are conjugated to a chemotherapeutic agent, immunotoxin, or radioactive particle. Conjugated MoAbs are designed to deliver a toxic agent to target cells, increasing its cytotoxic effect while minimizing side effects in nontargeted normal cells.3-5 An optimal target for MoAb therapy is an antigen highly expressed on the cellular surface membrane of the tumor cells with no presence as free protein in the plasma. It is necessary that, even when the antibody binds to the antigen, expression of the antigen remains at a high level on the target cells. On the other hand, the ability to induce immune responses with the production of human antimouse antibodies in the recipient, which block the therapeutic action of MoAbs, should be minimized. The development of humanized MoAbs, with the formation of chimeric proteins that have only a small antigen-binding mouse part and a large human constant Fc region, reduces their immunogenicity and prolongs their half-life. Since the introduction of rituximab, as the first targeted MoAb therapy for non-Hodgkin’s lymphomas (NHLs),6 MoAb therapy has become extensively used in patients with B-cell lymphoproliferative disorders.7,8 Rituximab (anti-CD20) and alemtuzumab (anti-CD52; Campath-1H, Ilex Pharmaceuticals, San Antonio, TX) have proven efficacy in different clinical trials.7,9-12 More reSeminars in Oncology, Vol 30, No 4 (August), 2003: pp 424-433
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cently, a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin (gemtuzumab-ozogamicin) has been approved for the treatment of CD33⫹ relapsed acute myeloid leukemia (AML).13,14 The purpose of this review is to discuss the main mechanisms of action of these three agents. ANTI-CD20 THERAPY
Rituximab is a chimeric humanized MoAb that contains a human IgG1 immunoglobulin constant region and a murine variable region specific for CD20. It binds to the surface antigen with the murine antibody part and stimulates the immune host mechanism through the human Fc portion.6 Rituximab has proven efficacy against a wide range of B-cell malignancies, including follicular lymphoma (FL), small lymphocytic lymphoma, marginal zone lymphoma, Waldenstro¨ m’s macroglobulinemia, mantle cell lymphoma, diffuse large cell lymphoma, and post-transplant lymphoproliferative disorders.7,8,15 CD20 is an integral transmembrane nonglycosylated hydrophobic phosphoprotein present on the surface of normal precursors and mature B cells; it remains expressed until terminal differentiation to plasma cells.16 CD20 is also present in the 90% of malignant B-cell NHLs and in about half of the cases with B-cell acute lymphoblastic leukemia; it is not expressed in neoplastic plasma cells of multiple myeloma.17 Importantly, CD20 expression is restricted to lymphoid B-cell lineage. The antigen is stable in the membrane of the B cell and does not shed, modulate, or internalize in response to antibody binding.6,18 The precise function of CD20 is still unknown, but it seems to play an important role in B-cell activation, differentiation, and growth.19-22 The CD20 cDNA predicts a tetraspan protein with intracellular NH2 and COOH termini and features of a membrane transporter or an ion channel.20 Cross-linking of CD20 results in modifications of B-cell growth and differentiation. However, the physiological CD20 ligands that would trigger similar responses have not been identified. The intracellular region of CD20 is rich in serine-threonine residues, contains multiple phosphorylation consensus sequences, and has been associated with the Src family kinases (Lyn, Fyn, and Lck), suggesting an implication of CD20 in transmembrane signaling pathways.23,24 It has been postulated that
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CD20 may be associated with lipid rafts.25-27 Lipid rafts are liquid-ordered membrane microdomains, enriched in sphingolipids and cholesterol, which are thought to function as platforms for signal transduction.28 In a recent study the association of CD20 to lipid rafts induced by rituximab produced a downregulation of the intracellular Lyn kinase.27 It has been postulated also that long-lasting perturbations of transmembrane signaling could contribute to progressive elimination of B cells.25,28 Mechanisms of Action of Rituximab Several mechanisms of action of rituximab have been proposed and analyzed in vitro, although the exact or predominant mechanism in vivo remains unknown. In vitro studies showed that rituximab is able to lyse CD20⫹ cells by inducing specific attack of tumor cells by immune cells through the Fc fixation (antibody dependent-cell mediated cytotoxicity [ADCC]) or by activation of the complement cascade (complement-dependent cytotoxicity [CDC]).29,30 In addition, rituximab induced apoptosis alone or in the presence of either goat antimouse IgG or Fc receptor-expressing cells.31,32 However, despite these in vitro data, whether the mechanism of action differs within compartments, and to what degree each of these mechanisms or other unrecognized mechanisms contributes to the destruction of tumor cells after treatment with rituximab, is unclear. ADCC and Rituximab ADCC is mediated by effector cells that express Fc receptors (FcR) such as natural killer (NK) and phagocytic cells. In vivo, FcR-expressing cells may infiltrate tumors and cross-link MoAb bound to the surface of tumor cells. ADCC and CDC are better mediated through the IgG1 constant region. The infusion in primates of an equivalent IgG4␥ version of rituximab is less effective in responses mediated by Fc and is unable to deplete B cells.33 Furthermore, there was a significant decrease in the antitumor effects of rituximab in Fc receptor– deficient mice.29 Three different classes of FcR for IgG (Fc␥R) have been described: Fc␥RI (CD64), Fc␥RII (CD32) and Fc␥RIII (CD16). Some FcR display a functional allelic polymorphism generating allotypes with different receptor affinities. Clinical responses of rituximab have been correlated with genetic polymorphisms identified on Fc␥RIIIa, an FcR present as a transmembrane mol-
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ecule on NK cells and monocytes. The polymorphism in Fc␥RIII, consisting of the presence in the amino acid position 158 of a valine (Val) or a phenylalanine (Phe), results in a different affinity for IgG1-Fc␥RIII binding. The Fc␥RIIIa-158 Val/ Val phenotype has greater affinity for human IgG1 than Fc␥RIIIa-158 Val/Phe or Phe/Phe.34 Among patients treated with rituximab alone for autoimmune diseases, those homozygous or heterozygous for the high-affinity allele achieve a greater depletion of B cells with the same plasma concentration of rituximab.35 In fact, 10-fold doses of rituximab are required in patients homozygous for the lowaffinity allele to obtain a similar reduction in B cells. In FL patients, the presence of homozygosity for Fc␥RIIIa-158Val is associated with a significantly higher objective response rate and a more frequent clearance of bcl-2 rearranged cells in both peripheral blood and bone marrow.36 Several groups have shown that incubation of tumor cells with rituximab and effector cells results in cellular lysis.30,37-39 Monocytes, NK cells, and neutrophils have been implicated in ADCC induced by rituximab,38 but the participation of different FcR in ADCC is not well understood. The recruitment of mononuclear cells seems to be mediated by CD16. Neutrophils normally express CD32 and CD16 and upon activation also express CD64. Tumor cell killing by neutrophils is more effectively triggered by CD64 and, as a result, is enhanced by granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colonystimulating factor (GM-CSF0.40,41 In addition, it has been described that phagocytosis of opsonized cells by macrophages is a process mediated by CD32; this process could be enhanced for cells less easily engulfed, such as chronic lymphocytic leukemia (CLL) cells, by the addition of GM-CSF.39 Finally, only NK cells are able to directly kill lymphoma cells in the presence of rituximab.38 In vitro assays of ADCC might not reflect what occurs during in vivo ADCC because ADCC experiments have been peformed in media depleted of complement, using effector:target cell ratios higher than expected in vivo30 and, normally, using allogeneic effector cells. Therefore, although these in vitro experiments show ADCC induced by rituximab, determination of its precise role in vivo requires additional investigation.
VILLAMOR, MONTSERRAT, AND COLOMER
CDC and Rituximab CDC is one of the mechanisms of action of rituximab. Through its human IgG1 Fc domain, rituximab can recruit serum proteins for CDC. The first component of the complement cascade, C1, is capable of binding the Fc portion of IgM and IgG molecules. C1 activation triggers the classical complement cascade, which leads to the formation of the membrane complex attack, which produces a direct lysis of the target cell. We and others have observed that CDC directly correlates with the expression of the CD20 antigen in malignant B cells, and the in vitro susceptibility to rituximab-mediated CDC depends primarily on CD20 protein expression (Fig 1).37,38,42 In fact, CDC is more rapidly and efficiently triggered by rituximab in cells with higher CD20 expression.37,42 It has been postulated that the lower rate of response to rituximab therapy in CLL patients compared with other lymphoproliferative disorders relates to the lower amounts of CD20 present on CLL cells. Therefore, the clinical differences of responses among patients could be related, at least in part, to CD20 expression. Deposition of complement proteins C337,43 and C937 is observed after in vitro treatment of B cells with rituximab in media containing complement, and colocalizes with rituximab.43 Whereas C3 deposition is observed independently of lytic responses and relates to CD20 expression, deposition of C9 and number of positive cells correlates with the degree of lysis.37 Moreover, the addition of anti-C3b(i) MoAb during in vitro experiments produces an increase in cell lysis.43 Furthermore, the administration of rituximab is followed by complement consumption.44 In a recent report analyzing gene expression in FL patients treated with rituximab, several complement proteins were among the genes differentially expressed between responders and nonresponders.45 The role of complement regulatory proteins in the modulation of rituximab efficacy has been addressed by different investigators.30,37,38,42,46 Several surface membrane proteins regulate the deposition of active complement proteins on cellular membrane to prevent cell lysis. Among these complement inhibitors, CD55 and CD59 seem to be the most important. No differences in the expression of CD59 molecules have been reported between normal B cells and malignant B cells from
MECHANISMS OF ACTION OF MONOCLONAL ANTIBODIES
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Fig 1. CDC is related to CD20 expression. (A) CD20 molecules per cell in responders (F) and nonresponders (E) in a series of patients with different B-cell lymphoproliferative disorders: CLL, FL, mantle cell lymphoma (MCL), and hairy cell leukemia (HCL). Response is mainly related to certain minimal amount of CD20 per cell. The gray bar highlights the zone where cells are heterogeneously sensible to rituximab. (B) Relationship between cell viability and CD20 expression after incubation with rituximab in the presence of complement. A direct correlation between CD20 molecules per cell and cytotoxicity was found.
different lymphoproliferative disorders,42 whereas low42 or high CD55 expression37 has been reported in CLL cells. Nevertheless, the in vitro susceptibility to rituximab-induced CDC could not be predicted by the levels of these proteins in CLL cells37,42 nor in vivo in FL47 and CLL patients.48 In contrast, using B-cell lines, a direct correlation between CDC and CD55 and CD59 has been reported by independent groups.38,49-51 Moreover, there is in vitro evidence that complement regulatory proteins play a role in the cytotoxic effect of rituximab (Fig 2). The blockage of CD55 and/or CD59 with specific antibodies sensitized cells to
Fig 2. CDC is increased after blockage with anti-CD55 and anti-CD59 monoclonal antibodies. Cells from five patients with CLL were incubated with rituximab and a complement source (Co) alone or with anti-CD55, anti-CD59 or both. Sensitization to rituximab was observed in all cases.
the cytotoxic effect of rituximab,37,42 indicating that these proteins, at least in vitro, may play an important role in rituximab’s action. Pretreatment with fludarabine52 or dexamethasone51 sensitizes cells to rituximab; it has been postulated for fludarabine that this is due to a decrease in the expression of CD55. All of these data suggest that analysis of CD55 and CD59 expression, in addition to that of CD20, might be useful to predict the response to rituximab in vivo. The ability of an anti-CD20 MoAb to recruit and activate complement is dependent not only on CD20 surface density or MoAb isotype, but also on the capacity of the antibody to translocate CD20 to lipid rafts.25 The mobility of CD20 molecules into lipid rafts would facilitate the engagement and activation of C1 and consequently the activation of complement cascade.25 CDC induced by rituximab results in cytoplasmic changes typical of apoptosis (ie, flow cytometry changes in forward side scatter (FSC) and side scatter (SSC) indicatives of cell shrinkage, exposure of phosphatidylserine residues, and decrease in the transmembrane mitochondrial potential). In contrast, no caspase activation and, consequently, no nuclear features of apoptosis are observed. Instead, a rapid and selective generation of reactive oxygen species (ROS) is detected after rituximab treatment. Finally, the use of ROS scavengers completely protects sensible cells to CDC induced by rituximab42 (Fig 3).
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Fig 3. Cellular modifications after incubation of malignant B cells with rituximab and complement (RⴙCo). (A) After 24 hours of in vitro treatment a loss of membrane integrity (propidium iodide–positive cells) an exposure of phosphatidylserine (annexin V–positive cells) is observed. (B) Loss of mitochondrial potential (DIOC6-negative cells) and generation of reactive oxygen species (ROS) (dihydroethidine-positive cells) in RⴙCo-treated cells. (C) Absence of caspase activation. No increase in cells expressing the active form of caspase 3 after RⴙCo treatment. (D) Incubation of cells with a ROS scavenger (N-acetyl-cysteine, NAC) protects cells from the RⴙCo effect. (E) Rapid generation of ROS after addition of a source of Co in cells pretreated for 10 minutes with rituximab.
Apoptosis and Rituximab Induction of apoptosis is another mechanism of action described for rituximab treatment. Apoptosis is observed after cross-linking,32,49 and some reports indicate that rituximab alone can induce apoptosis in B-cell lymphoma cell lines.31,38 The apoptotic process implicates the mitochondrial release of cytochrome c, loss of transmembrane mitochondrial potential, and activation of caspase-9 and the effector caspase-3.24,53 Although crosslinking of rituximab may lead to mitochondrial changes and caspase activation, these events are not necessary for relay of the apoptotic signal. It has been postulated that the apoptosis induced by anti-CD20 antibody in B-cell lines is dependent on increases in intracellular calcium, since apoptosis is inhibited using some extracellular calcium chelators.32 In patients with CLL, tumor cell clearance has been related to induction of apoptosis involving
the mithochondrial pathway and activation of caspases. After rituximab infusion, active forms of caspase-9, caspase-3, and poly-adenosine diphosphate ribose polymerase (PARP) are detected in peripheral blood of a proportion of these patients, followed by a decrease of lymphocyte counts. Furthermore, a significant downregulation of the antiapoptotic proteins XIAP and Mcl-1 is also observed.54 Recently, the Mcl-1/Bax ratio has been related to the clinical response in CLL patients.48 In some B-cell lines, downregulation of the antiapoptotic protein bcl-2 through interleukin-10 inhibition has been reported after rituximab incubation, rendering cells more sensitive to cytotoxic agents.55 These results suggest that the cytotoxic effect observed in vivo after rituximab treatment induces apoptosis, using a final pathway similar to chemotherapeutic agents.56 The differences in induction of apoptosis within anti-CD20 antibodies could be due to their differ-
MECHANISMS OF ACTION OF MONOCLONAL ANTIBODIES
ent ability to segregate into rafts. Anti-B1, or tositumomab, is a anti-CD20 IgG2a that seems to bind an overlapping epitope of rituximab with a different affinity.49 In opposition to rituximab, tositumomab has shown significant antitumor activity and is able to induce apoptosis on Ramos cells in the absence of cross-linking.31,49,57-59 Cross-linking of tositumomab did not further increase cell cytotoxicity.49 These two antibodies differ also in their ability to induce CDC, which has been related with translocation to lipid rafts.25 Finally, dimerization of rituximab may also influence its ability to induce cell death. Thus, it has been observed that homodimeric rituximab dissociates more slowly than the monomeric form and only homodimers are able to induce apoptosis in cell lines.31 This could explain in part the differences observed in in vitro experiments. Mechanisms of Resistance to Rituximab In normal individuals, CD20 is not detected as a free protein in plasma. This situation is an ideal scenario for anti-CD20 MoAb therapy. Nevertheless, it has been recently shown that CLL patients may have significant amounts of circulating CD20. The presence of plasma CD20 is not due to active shedding or breakdown of cells and is supposed to circulate in large complexes or as fragments of cell membrane. The circulating CD20 levels vary among patients and seem to be associated with unfavorable clinical and biological parameters without correlation with lymphocyte counts. More importantly, circulating CD20 acts as a competitor for cellular CD20 in rituximab binding.60 Therefore, the presence of circulating CD20 might explain some treatment failures. A number of patients previously treated with rituximab who subsequently relapsed may display CD20⫺ tumors.61,62 The exact incidence of this phenomenon is unknown. In addition, a transient presence of malignant CD20⫺ B cells or modification in cell phenotype has been observed after rituximab treatment.63,64 This may explain some cases of resistance to re-treatment with rituximab. ANTI-CD52 THERAPY
CD52 antigen is a glycoprotein with an apparent molecular mass of 21 to 28 kd. It comprises a short peptide sequence of 12 amino acids and a large complex N-linked oligosaccharide that is attached to the membrane by a glycosylphosphati-
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dylinositol (GPI)-anchoring structure and is constitutively expressed in membrane lipid rafts. CD52 antigen is highly expressed on normal and neoplastic lymphocytes, monocytes, and macrophages, but not on erythrocytes or stem cells, and does not modulate.65 The antigen is also expressed on a large proportion of lymphoid cell malignancies. The function of CD52 remains unknown. Alemtuzumab (Campath-1H) is a recombinant DNA-derived humanized MoAb directed against the cell surface glycoprotein CD52. The alemtuzumab antibody is an IgG1 with human variable framework and constant regions, and CDR from a murine MoAb.66 Alemtuzumab treatment resulted in complete depletion of CD52⫹ cells, including B and T lymphocytes, monocytes, and NK cells from the peripheral blood. Alemtuzumab is increasingly used in the treatment of lymphoid malignancies, autoimmune diseases, and bone marrow transplantation, although it has a significant toxicity. Also, given its ability to deplete T cells, alemtuzumab has been used to prevent graft-versus-host disease following transplantation.67 Mechanism of Action of Alemtuzumab The mechanism of action of alemtuzumab has not been well established and this agent has been less extensively studied than rituximab. It has been proposed that CDC, ADCC, and apoptosis68-70 could mediate cell lysis. Cross-linking CD52 with the humanized MoAb alemtuzumab induces growth inhibition and apoptosis, the degree of which is related to the antigen density on the cell.68 In addition, the in vivo response to alemtuzumab has been correlated with CD52 expression on malignant cells.71 The mechanism of induction of proliferation arrest and apoptosis is not clear, but it could be mediated by tyrosine phosphorylation.68 The first dose-reaction observed after alemtuzumab was due to CD16 cross-linking of NK cells that release inflammatory cytokines including tumor necrosis factor-␣, interleukin-6, and interleukin-1.70 Despite the fact that they both carry the same human IgG1 Fc region, it has been suggested that rituximab and alemtuzumab differ significantly in their ability to activate human complement, with rituximab being a stronger activator than alemtuzumab.46 Complement activation is not completely necessary for depletion of CD52⫹ cells in vivo72 and cell lysis mediated by ADCC requires
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1,000-fold less antibody than CDC.73 This may be due to the secondary structure of each MoAb and its spatial relationship relative to the cell surface, which may affect its capacity to bind to C1 and activate the classic complement cascade.74 Mechanism of Resistance to Anti-CD52 Therapy After alemtuzumab treatment, the emergence of lymphocytes deficient in GPI-anchored proteins has been described.68,75-77 The degree of GPI-deficient cells and the time required to recover differs between B and T lymphocytes, with T cells being more frequently CD52⫺ than B cells.77,78 The phenotype of these CD52⫺ B and T cells closely resembles that of lymphocytes from paroxysmal nocturnal hemoglobinuria (PNH) patients, in which the first step of the GPI pathway is blocked due to mutations in the PIG-A gene. However, the molecular mechanism of the appearance of PNHlike clone after alemtuzumab is not well established. In CLL patients, a selection of cells carrying a PIG-A mutation present at low levels before treatment, and a decrease in mRNA PIG-A gene, has been described as a mechanism of emergence of PNH-like cells.75,76 Finally, some cases of CD52⫺ relapses in patients treated with alemtuzumab have been reported.79,80 ANTI-CD33 THERAPY
CD33, a myeloid differentiation antigen, is a member of the immunoglobulin superfamily and is a membrane-bound glycoprotein of 67 kd. CD33 is expressed by human monocytes, promyelocytes, myeloid blasts, and occasionally by acute lymphoblastic leukemias; it is absent from normal hematopoietic stem cells and nonhematopoietic tissues.81 Although it has been shown that the CD33 antigen is a sialic acid– binding receptor,82,83 its function on myeloid cells remains to be established. Gemtuzumab-ozogamicin (Mylotarg, WyethAyerst Laboratories, St Davids, PA) is a humanized IgG4 MoAb (hP67.6) against CD33 linked to a calicheamicin derivative (N-acetyl ␥1 calicheamicin).14 This molecule is a member of the enediyne family of antitumor antibiotics, which are more cytotoxic than other clinically used anticancer agents. Gemtuzumab-ozogamicin is rapidly internalized after binding to its target, followed by the release of the potent antitumor calicheamicin derivative. This compound-induced
double-stranded DNA breaks resulting in apoptosis.84 In this case, the mechanism of action may be different from that of rituximab and alemtuzumab. Gemtuzumab-ozogamicin induces DNA damage followed by cell cycle arrest in order to allow the cell to repair DNA or enter into apoptosis. It has been observed that within 3 to 6 hours after infusion, a near complete saturation of CD33 antigenic sites by is reached for AML blasts, monocytes, and granulocytes. After binding to CD33 antigen, gemtuzumab-ozogamicin is rapidly internalized and induces a dose-dependent apoptosis in myeloid cells in vitro.85 The response of AML cell lines to gemtuzumab-ozogamicin is heterogeneous. In some cell lines, gemtuzumab-ozogamicin induces an arrest in the G2 phase of the cell cycle, while other cell lines are blocked in G2 but rapidly undergo apoptosis and still others are resistant to gemtuzumab-ozogamicin. These different responses do not correlate with the levels of expression of CD33, and it has been proposed that the ATM/ATR-chk1/chk2 pathway is implicated in the cell cycle response to gemtuzumab-ozogamicin.86 No correlations between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including gemtuzumab-ozogamicin have been reported.87 Moreover, it has been described that residual marrow leukemia after gemtuzumab-ozogamicin treatment and failure to achieve complete or partial remission correlated highly with blast cells P-glycoprotein functions and with low in vitro drug-induced apoptosis.88 CONCLUSIONS
The use of MoAbs as targeted therapy for the treatment of hematologic malignancies has been increasing over the last few years. The mechanisms of action of these MoAbs are being increasingly better known. These include induction of apoptosis, ADCC, and CDC. The relative contribution of each mechanism and the role of genetic variability of the patients in the response to therapy remain to be determined. Advances in the understanding of the genetics of tumors and further investigation on the mechanisms of action of MoAbs will help in designing more specific and effective therapies.
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