r eVi eW
`
`Cd20-targeted therapy: a breakthrough in the
`treatment of non-Hodgkin’s lymphoma
`
`T. van Meerten1, A. Hagenbeek1,2*
`
`1Department of Haematology, University Medical Centre Utrecht, Utrecht, 2Department of
`Haematology, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands,
`tel.: +31 (0)20-566 91 11, e-mail: a.hagenbeek@amc.uva.nl
`
`a b s t r aC t
`
`targeting the Cd20 antigen on b lymphocytes with the
`monoclonal antibody rituximab has greatly improved
`the outcome of patients with b-cell malignancies.
`despite the success of rituximab, resistance occurs in
`about half of the patients, resulting in non-response
`to treatment or early relapse of the original disease. a
`better understanding of the mechanism of rituximab
`resistance has lead to the development of novel, improved
`anti-Cd20 antibodies. this review describes the
`development of Cd20-targeted therapy from its historical
`background towards the next generation of anti-Cd20
`monoclonal antibodies and explains new strategies to
`overcome resistance.
`
`Rituximab eliminates CD20-positive cells mainly through
`three different mechanisms: complement-dependent
`cytotoxicity (CDC), antibody-dependent cellular cytotoxicity
`(ADCC) and the induction of apoptosis. Resistance to
`rituximab can be lymphoma-related or host-related. The
`preference for one of these mechanisms depends on
`the patient-specific microenvironment of the lymphoma.
`Based on the physiology of these factors, novel anti-CD20
`antibodies are being developed.
`This article reviews the development of CD20 targeting
`from its historical background towards the next generation
`of anti-CD20 monoclonal antibodies and explains the new
`strategies to overcome resistance.
`
`
`K e y Wo r d s
`
`H U m a n C d2 0
`
`Anti-CD20-therapy, CD20, non-Hodgkin’s lymphoma,
`rituximab
`
`Expression of the human CD20 molecule is restricted
`to B-cell precursors and mature B cells ( figure 1). CD20
`
`i n t r o d U C t i o n
`
`figure 1. The human CD20 molecule
`
`The goal of CD20-targeted therapy is to kill B lymphocytes
`by the use of monoclonal antibodies (MoAb) against the
`B-cell specific human CD20 molecule. Clinical success
`started by targeting non-Hodgkin’s lymphoma (NHL)
`with rituximab, a chimeric anti-CD20 MoAb. The use
`of rituximab as a single agent or as an addition to
`chemotherapy in NHL patients can be considered as one
`of the most successful and worldwide accepted forms of
`immunotherapy so far.
`However, despite its success, resistance occurs in about half
`of the NHL patients, resulting in non-response to treatment
`or early relapse of the original disease.
`
`© 2009 Van Zuiden Communications B.V. All rights reserved.
`
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`expression is lost upon differentiation of the B cells towards
`plasma cells.1-3 As shown in figure 2, CD20 is expressed
`within key B-cell development stages that give rise to B-cell
`NHL and chronic lymphocytic leukaemia (CLL).
`CD20 is an ideal target for antibody-mediated therapy
`because CD20 is not expressed in haematopoetic stem
`cell B cells, so that the B-cell haematopoiesis and other cell
`lineages are not in danger. Moreover, CD20 is not expressed
`on plasma cells, which means that antibody therapy will
`not significantly decrease the immunoglobulin production
`against pathogens. Other advantages of targeting CD20
`are that CD20 does not circulate in the plasma,4 is not shed
`from the cell surface5 and is not internalised6 after antibody
`binding. Although CD20 is the most frequently antibody-
`targeted antigen in general, its exact function is still
`unknown. Actually, the CD20 antigen was discovered
`through generation of the first anti-CD20 monoclonal
`antibody. Balb/c mice were immunised with Burkitt’s
`lymphoma cells and a new antibody was formed, called
`anti-B1, which recognised CD20.1 Still no natural ligand
`is known for CD20 and our current understanding of
`the function of the CD20 molecule comes from ligation
`with different antibodies to CD20.7-10 These experiments
`suggest that CD20 functions as a B-cell activating or
`proliferation molecule. Different antibodies have shown
`effects on B-cell proliferation, and some were able to
`block B-cell growth (reviewed in Deans et al.).7 In general,
`ligation of CD20 with most antibodies (type I anti-CD20
`MoAb) leads to the formation of signalling platforms
`(lipid rafts) and eventually to calcium flux and activation
`of caspase-3.11 The formation of these signalling platforms
`and the downstream signalling cascade is probably in
`conjunction with the signalling potential of the B-cell
`receptor (BCR).12
`
`d e V e l o P m e n t o f t H e a n t i - C d2 0
`a n t i b o d y r i t U x i m a b
`
`The first monoclonal antibody that recognised CD20, the
`murine anti-CD20 B1, was generated in 1980.1 Because of
`their potential in the treatment of B-cell disorders, in the years
`thereafter anti-CD20 antibodies were genetically engineered
`for clinical application. In 1997, rituximab (MabThera®,
`Rituxan®) was the first MoAb approved specifically for the
`treatment of patients with relapsed or refractory CD20-positive
`low-grade (follicular) non-Hodgkin’s lymphoma. Rituximab
`is a chimeric anti-CD20 antibody that is engineered as
`follows: the light and heavy chain variable regions from the
`murine 2B8 anti-CD20 antibody (IDEC-2B8), generated by
`immunising mice with a CD20-positive human lymphoma,
`are amplified by polymerase chain reaction and inserted into
`a cDNA mammalian chimeric antibody expression vector,
`which also contains the neomycin phosphotransferase gene
`(NEO). This vector is electroporated into Chinese hamster
`ovary (CHO) cells and under antibiotic pressure the cells
`stably secrete Ig levels.13 The resulting chimeric antibody is
`purified and consists of a human kappa constant region, a
`human IgG Fc portion (IgG1), and a murine variable region,
`recognising the human CD20 protein.13
`
`a C t i o n m e C H a n i s m s o f r i t U x i m a b
`
`Upon ligation of CD20, rituximab triggers different
`effector mechanisms. Many in vitro and in vivo studies have
`been conducted to explore the most important one. In vitro,
`it is well established that there are three main modes of
`action of rituximab: 1) induction of apoptosis 2) CDC and
`3) ADCC, as described below ( figure 3).
`
`figure 2. CD20 expression in B-cell development
`
`CML
`
`Precursor
`B-cell acute
`leukaemias
`
`B-cell lymphomas
`CLL
`
`CD20+
`
`Myeloma
`
`Haema to-
`poietic stem
`cell
`
`Lymphoid
`stem cell
`
`Pro-B cell
`
`Pre-B cell
`
`Immature
`B cell
`
`Mature
`B cell
`
`Activated
`B cell
`
`Memory
`B cell
`
`Plasma cell
`
`Bone marrow
`
`Blood, lymph
`
`the Cd20 antigen is first expressed during early pre-b-cell development and is lost during terminal plasma cell differentiation.
`
`Van Meerten, et al. CD20-targeted therapy.
`
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`figure 3. The three main mechanisms of action of rituximab
`
`Direct induction of apoptosis
`
`Ligation of rituximab to CD20
`
`Complement-dependent cytotoxicity
`
`Complement-
`binding
`
`Invitation/triggering of signal
`transduction pathways
`
`Assembly of
`MAC
`
`Antibody-dependent cellular cytotoxicity
`
`FcR-
`binding
`
`Natural killer
`cell macrophage
`granulocyte
`
`Killing via engulfment of release
`of perforin and granzyme B
`
`after rituximab ligation to Cd20, three different effector mechanisms can be activated. 1) direct induction of apoptosis may be caused by the
`activation of the death receptor pathway or the mitochondrial pathway by modulation of the bcl-2 gene family. but apoptosis-induced death can also
`bypass the mitochondrial pathway or caspases. 2) the fc portion of the igG1 tail of rituximab is able to bind C1q, the first component of the classical
`complement pathway. binding of C1q triggers a proteolytic cascade, resulting in accumulation of C3b. C3b molecules act as opsonins for phagocyting
`cells but also bind to the C3 convertase to form a C5 convertase, leading to the generation of the membrane attack complex (maC), which kills the
`cell. 3) the fc portion is also recognised by cells expressing the fc receptors (nK cells, granulocytes and macrophages). Cell killing is mediated by
`phagocytosis or the release of cytotoxic granules.
`
`rituximab and apoptosis
`Data concerning the mechanism of the apoptotic effect
`of rituximab are conflicting. Different groups obtained
`different results, even if they used similar target cell
`lines.7,14,15 It has been suggested that one of the late
`apoptotic pathways, caspase-3, is activated.16 However,
`others documented that the apoptotic pathways are caspase
`or Fas ligand/ Fas death pathway and mitochondria
`independent, and do not require lipid raft formation.14,17
`Hyper-crosslinking of rituximab, either by a secondary
`antibody or by Fc bearing effector cells, generally increased
`the apoptosis.7 An important observation is that within
`a treated cell population not all cells uniformly undergo
`apoptosis. This is the current focus of many groups that
`study rituximab resistance.
`
`rituximab and CdC
`The Fc portion of rituximab is able to trigger the
`classic complement system, resulting in CDC. In vitro,
`C1q is bound efficiently by rituximab.13,18 and simple
`CDC assays demonstrate that complement activation
`induces cell kill.15,19,20 Rituximab-induced CDC has a
`variable degree of efficiency, which has been associated
`
`with expression of complement regulatory proteins
`(CRP) CD55 and CD59.15,20,21 Whereas CD20 expression
`level has been suggested to be an important predictor
`of clinical CDC efficiency, several studies show
`contradictory results and no clear evidence for this
`relationship.15,21-23
`
`rituximab and adCC
`ADCC is mediated by effector cells expressing FcγRI
`(CD64), FcγRII (CD32) or FcγRIII (CD16). Effector cells,
`such as NK cells, granulocytes or macrophages, are able
`to recognise the Fc portion of rituximab, and kill the
`ligated cells by phagocytosis or the release of cytotoxic
`granules.13,15,24,25 For ADCC, it has been demonstrated that
`the efficacy depends on polymorphisms of the effector
`cells.25,26
`
`In the in vitro studies it was possible to investigate the
`mechanisms of rituximab separately, but this is more
`complex for in vivo studies. In several murine studies it
`was attempted to clarify the importance of each effector
`mechanism. Elegant mouse models using FcγR-deficient
`mice pointed out that clearing of CD20-expressing cells
`
`Van Meerten, et al. CD20-targeted therapy.
`
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`was FcγR dependent for a panel of murine anti-CD20
`MoAb27 and rituximab.28 Other groups demonstrated
`that complement was responsible for CD20-positive
`tumour clearance by rituximab.9,29,30 However, there is no
`agreement in the literature about the dominance of one
`particular in vivo effector mechanism.
`Also, some evidence concerning the mechanism
`of rituximab has been obtained in patients. One of
`the infusion-related side effects of rituximab is the
`complement consumption after administration,31,32
`indirectly confirming CDC. On the other hand, clinical
`responses have been correlated to polymorphisms in the
`FcγRIIIA gene,26,33,34 indirectly confirming ADCC. In
`addition, a significant direct effect of rituximab cell kill by
`activating caspase-3 was demonstrated in vivo in patients
`with chronic lymphocytic leukaemia (CLL).16
`
`C l i n iC a l a P P l iC a t i o n o f
`r i t U x i m a b
`
`The first phase I trial in humans with rituximab as a
`single agent was conducted in 1993 for patients with
`relapsed low-grade B-cell lymphoma.35 Within five
`single-agent trials, no severe toxicities were found and
`only infusion-related adverse events occurred within
`the first hours, in particular after the first infusion. The
`most common side effects were chills, fever, nausea,
`fatigue, headache and angio-oedema.36 Several phase
`II and III trials studied the optimal schedules and
`dosing with or without chemotherapy, biologicals, and
`radiotherapy.36 After approval in the USA in 1997 and
`in Europe in 1998, rituximab was included in the
`standard treatment of NHL. Rituximab works very
`efficiently in combination with chemotherapy. For diffuse
`large B-cell lymphoma (DLBCL), follicular lymphoma
`and mantle cell lymphoma, inclusion of rituximab in
`standard chemotherapy regimens significantly improved
`patients outcome with or without pretreatment37-46
`and is accepted as a standard first-line therapy for
`CD20-positive lymphomas. Moreover, if patients with
`low-grade lymphoma respond to single-agent rituximab
`therapy, progression-free survival and overall survival
`are substantially prolonged with scheduled maintenance
`treatment.47,48 In patients who achieved complete or
`partial remission after the combination of chemotherapy
`and rituximab, maintenance with rituximab increased
`the overall and progression-free survival.46-49 In addition,
`rituximab maintenance in patients treated after standard
`chemotherapy significantly increased the three-year
`progression-free survival from 33 to 68%.49,50
`The therapeutic effect of rituximab, through the
`depletion of B cells, has also proven to be successful
`for patients with B-cell related autoimmune diseases.
`
`Examples are rheumatoid arthritis, autoimmune
`thrombocytopenic purpura, inflammatory skin diseases
`and pemphigus, systemic lupus erythematosus and
`other forms of vasculitis, diabetes, neurological
`diseases such as chronic inflammatory demyelinating
`polyneuropathy (CIDP) and multiple sclerosis51-53 and
`chronic graft versus host disease after allogeneic stem
`cell transplantation.54-57
`
`r i t U x i m a b r e s i s t a n C e
`
`However, despite the success story, resistance of lymphoma
`B cells towards rituximab is observed in about half of the
`patients in the course of prolonged treatment. The precise
`mechanism of resistance to rituximab is unknown.
`Resistance may be tumour-related or host-related.
`Tumour-related resistance could be the lower number
`of CD20 molecules per cell, the increased expression
`of complement regulatory proteins or expression
`of antiapoptotic genes. Host-related resistance is
`determined by polymorphisms in the FcγRIIIA gene
`effector cells.26,33,34 The cellular microenvironment
`probably contributes to the dominant effector and
`resistance mechanism of rituximab.58 There is a
`difference in the extent of B cell depletion in peripheral
`blood, lymph nodes and spleen. Also, within the lymph
`node there is a differential susceptibility of different
`B-cell subsets to MoAb treatment.58,59 In a human-CD20
`transgenic mouse model, Gong and colleagues
`demonstrated that circulating B cells are depleted
`mainly through the macrophages of the reticulo-
`endothelial system, while B cells within the marginal
`zone compartment in lymph nodes depend on CDC
`rather than FcγR-mediated depletion. In fact, marginal
`zone B cells that are trafficking from the marginal zone
`to the vasculature make them susceptible for depletion
`with MoAbs. B cells residing in the lymphoid tissues
`depend on the vasculature for accessibility of effector
`cells.58 In addition, in some lymph node compartments
`(germinal centres) B cells receive additional survival
`signals. Exposure to these signals makes these cells
`less sensitive to anti-CD20.58,59 The significance of the
`microenvironment in rituximab-induced cell death is
`also indirectly observed by differential responses to
`rituximab therapy in different subtypes of CD20-positive
`lymphomas (which have unique microenvironments),
`and is furthermore supported by the observation that
`molecular remissions in the blood and bone marrow
`induced by rituximab can occur in the setting of
`progressive nodal disease. More knowledge on and/or
`manipulation of the microenvironment may lead to
`developing a means to decrease or overcome rituximab
`resistance.
`
`Van Meerten, et al. CD20-targeted therapy.
`
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`Several attempts have been made to improve rituximab
`efficacy and thereby to overcome resistance. For example,
`down-regulation of the antiapoptotic bcl-2 gene by
`antisense oligonucleotides may enhance the apoptotic
`effect of rituximab.60 Other attempts were made to improve
`ADCC by immunostimulatory molecules such as IL-2,
`IL12, IL15 or CpG sequences.61-63 or improving CDC by
`down-regulation of complement regulatory proteins, but
`with limited success.20,22,23
`
`More promising is the next generation of monoclonal
`anti-CD20 antibodies ( figure 4). In recent years, different
`murine, humanised and completely human anti-CD20
`MoAbs have been developed (for nomenclature see table 1).
`These antibodies may bind to a different epitope or induce
`a specific mechanism of action. Another way to classify
`these antibodies is the ability to translocate CD20 into the
`lipid rafts. Anti-CD20 antibodies are either type I or type
`II (see also table 2).
`Type I antibodies relocate CD20 molecules into lipid
`microdomains, which can act as signalling platforms. These
`antibodies are potent CDC inducers. Rituximab belongs to
`the type I antibodies. Type II antibodies do not redistribute
`CD20 into signalling platforms and do not induce CDC.
`However, type II antibodies promote strong homotypic
`adhesion and have a strong induction of direct cell death.
`
`table 1. Nomenclature of therapeutic monoclonal
`antibodies
`
`suffix to generic name
`-omab
`-amab
`-emab
`-imab
`-ximab
`-zumab
`-umab
`
`origin
`Murine
`Rat
`Hamster
`Primate
`Chimeric
`Humanised
`Human
`
`Table 3 gives an overview of new anti-CD20 MoAbs
`in comparison with rituximab. They are summarised
`below.
`
`H U m a n a n t i b o d y ( t y P e i )
`
`ofatumumab
`Ofatumumab is a completely human anti-CD20 antibody.
`Ofatumumab, a type I MoAb, is generated in human
`immunoglobulin transgenic mice. Compared with
`rituximab, it binds a different epitope on the CD20
`molecule and has a slower off rate. Ofatumumab binds
`the small 7-mer loop of the human CD20 molecule, which
`is in a closer proximity to the cell membrane than the
`binding site of rituximab, which binds the larger 44-mer
`loop. This is probably the most important reason why
`ofatumumab is more potent than rituximab in inducing
`complement.10,64 First clinical data with ofatumumab
`showed safe application and responses to therapy in
`
`table 2. Differences between type I and II anti-CD20
`monoclonal antibodies
`
`type ii moabs
`type i moabs
`Localise CD20 to lipid rafts Do not localise CD20 to lipid rafts
`High CDC
`Low CDC
`ADCC activity
`ADCC activity
`Full number of binding
`Half number of binding sites/B-cell
`sites/B-cell
`Weak homotypic
`aggregation
`Weak direct cell death
`induction
`Examples:
`Rituximab
`Ocrelizumab
`Ofatumumab
`Veltuzumab
`AME-133
`PRO131921
`
`Examples:
`GA101
`B1 (Tositumomab)
`
`Strong homotypic aggregation
`
`Strong direct cell death induction
`
`figure 4. Development of monoclonal antibodies recognising CD20
`
`Variable region (recognises CD20)
`
`Constant κ region
`
`Human IgG1-Fc region
`
`Murine
`
`Chimeric
`
`Humanised
`
`Human
`
`murine anti-Cd20 mabs are generated by immunisation of mice with Cd20-positive cells. in chimeric antibodies, the 30% that is of murine origin
`is the variable region that recognises the Cd20 antigen. the variable regions are cloned into a chimeric antibody expression vector, resulting in an
`antibody which contains the constant κ region and the igG1-fc region of human origin. for humanised antibodies, also with cloning techniques,
`the variable region is modified to be more human. Humanised antibodies contain complementary-determining regions of murine origin, which
`recognise the Cd20 antigen. only 10% of the antibody is of murine origin. Human anti-Cd20 mabs are derived from human immunoglobulin
`transgenic mice. the latter antibodies are likely to be non-immunogenic in men.
`
`Van Meerten, et al. CD20-targeted therapy.
`
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`table 3. Second- and third-generation anti-CD20 antibodies
`
`antibody
`
`antibody specificity
`
`activity (compared with
`rituximab)
`
`additional features
`(compared with rituximab)
`
`Ofatumumab
`
`type isotype Cdr
`I
`IgG1
`Human
`
`CdC adCC apoptosis
`+++
`=
`=
`
`Ocrelizumab
`
`PRO131921
`
`Veltuzumab
`
`AME-133
`
`Tositumomab
`
`GA-101
`
`I
`
`I
`
`I
`
`I
`
`II
`
`II
`
`IgG1
`
`Humanised
`
`IgG1
`
`Humanised
`
`=
`
`=
`
`IgG1
`
`Humanised
`
`=/+
`
`IgG1
`
`Humanised
`
`IgG2A Murine
`
`IgG1
`
`Humanised
`
`=
`
`-
`
`-
`
`=/+
`
`++
`
`=
`
`+
`
`=
`
`=
`
`=
`
`=
`
`=
`
`++
`
`+++
`
`+++
`
`•
`
`Binds the small extracel-
`lular part of CD20
`•
`Completely human
`•
`Slower off-rate
`Binds a different but over-
`lapping epitope compared
`with rituximab
`Enhanced affinity for
`FcγRII
`Slower off-rate
`
`Enhanced affinity for
`CD20
`Bound to radio-isotopes
`
`•
`•
`
`γRII
`High affinity for Fc
`Strong induction of
`apoptosis
`
`Clinical trials
`(www.clinicaltrials.
`gov)
`
`Phase I/II: RA, FL,
`CLL, WM, RRMS.
`Phase III: CLL, FL,
`DLBCL
`Phase I, II, III: RA
`Phase III: SLE
`Phase II: RRMS
`Phase I/II: CLL,
`NHL
`Phase I/II: CLL,
`NHL, ITP
`Phase I/II: NHL
`
`references
`
`10, 64-66
`
`59, 67, 68
`
`69
`
`70-74
`
`75, 76
`
`Bound to radio-
`isotopes: NHL
`Phase I/II: NHL
`
`9, 77, 79
`
`79-81
`
`Cdr = complementary determining regions; igG = immunoglobulin; CdC = complement-dependent cytotoxicity; adCC = antibody-dependent
`cellular cytotoxicity; ra = rheumatoid arthritis; fl = follicular lymphoma; Cll = chronic lymphocytic leukaemia; rrms = relapsing remitting
`multiple sclerosis; Wm = Waldenstrom’s macroglobulinaemia; dlbCl = diffuse large b cell lymphoma; sc = subcutaneous; sle = systemic lupus
`erythematosus; itP = idiopathic autoimmune thrombocytopenic purpura; nHl = non-Hodgkin’s lymphoma.
`
`rituximab-resistant patients.65,66 Clinical responses to
`ofatumumab in a phase I/II trial are promising. In this trial
`in patients with follicular lymphoma, previously treated
`with rituximab, clinical responses with ofatumumab
`were up to 63% with a median time to progression of
`32.8 months.66 Ofatumumab is currently being used in
`different phase III trials.
`
`H U m a n i s e d a n t i b o d i e s ( t y P e i )
`
`ocrelizumab (Pro70769 or rhuH27)
`Ocrelizumab
`is derived from the murine 2H7
`anti-CD20 antibody and humanised with recombination
`techniques. Ocrelizumab is a type I MoAb and has an
`IgG1 isotype. Compared with rituximab, ocrelizumab
`binds a different, but overlapping epitope on the large
`extracellular part of CD20 and shows a two to fivefold
`increased ADCC and three to fivefold decreased CDC,
`which might lessen infusion-related reactions.67 In a phase
`I/II study, ocrelizumab was administered to rituximab-
`pretreated patients with relapsed/refractory follicular NHL.
`Ocrelizumab was well tolerated and showed a response
`rate of 36%.67
`In cynomolgus monkeys ocrelizumab was shown to have
`the same B-cell depleting capability as rituximab.59 In the
`ACTION study group, ocrelizumab in combination with
`methotrexate was studied in a phase I/II trial in the treatment
`of RA. Over a 72-week follow-up ocrelizumab appeared to be
`safe with minimal immunogenicity and longer duration of
`
`the B-cell depletion.68 Currently, ocrelizumab is undergoing
`phase III clinical trials for RA and lupus nephritis, and phase
`II trials for multiple sclerosis.
`Modification of ocrelizumab resulted in an antibody
`with improved binding to FCγRIIIa and possibly a better
`ADCC. This version of ocrelizumab, called PRO131921, is
`studied in a phase I/II trial in the treatment of relapsed or
`refractory CLL and indolent NHL.69
`
`Veltuzumab (ha20, immU-106)
`Veltuzumab is a type I, humanised IgG1 MoAb generated
`by using the same human framework as epratuzumab
`(humanised anti-CD22). The complementary determining
`regions (CDR) were taken from the parental murine A20.
`Compared with rituximab there is a single amino acid
`difference in CDR3-VH. For this reason, veltuzumab has a
`slower off rate and improved in vivo activity.70 In vitro, the
`three main mechanisms of action are similar to rituximab.71
`The first clinical studies have shown favourable safety and
`efficacy results in NHL patients with lower doses and less
`administrations of antibody.72-74 Overall response rate in
`rituximab-pretreated patient with refractory or relapsed
`NHL was 44%.74 In a phase I/II study, subcutaneous
`administration of veltuzumab in NHL and CLL is being
`studied and also a phase I study is ongoing for the treatment
`of autoimmune thrombocytopenic purpura.
`
`ame-133 (ly2469298)
`The production of this antibody is based on the fact that
`there is a strong correlation between FcγRIII (CD16)
`
`Van Meerten, et al. CD20-targeted therapy.
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`polymorphisms and MoAb efficacy.75,76 AME-133 is a type
`I, humanised IgG1 MoAb. It consists of a human germline
`framework region in which CDRs were inserted. CDRs were
`synthesised using a mutagenesis procedure by targeted
`insertion of synthetic oligonucleotide pools and their
`selection is based on enhanced MoAb affinity for CD20.
`In addition, the Fc region was also modified by targeting
`the constant region with synthetic oligonucleotides.
`This resulted in an antibody with enhanced affinity for
`human FcγRIII and with an enhanced ADCC activity as
`compared with rituximab. The clinical efficacy of AME-133
`is currently being studied in a phase I/II trial for the
`treatment of NHL. No clinical data are available yet.
`
`m U r i n e a n t i b o d y ( t y P e i i )
`
`tositumomab
`Tositumomab (B1) is a murine IgG2a lambda MoAb.
`Ionising radiation therapy with covalently linked Iodine-131
`to tositumomab is successfully used for the treatment
`of patients with follicular and transformed NHL who
`failed or relapsed from prior rituximab treatment and
`standard chemotherapy.77 Without the conjugation of
`an ionising agent, tositumomab also has a direct toxic
`effect. In vitro data show that tositumomab is far more
`efficient in inducing apoptosis and murine models show
`that tositumomab can prolong the survival of mice
`injected with Daudi lymphoma cells, in the absence of
`complement.9 In addition, preclinical studies demonstrate
`that tositumomab is more efficient in depleting B
`cells than rituximab.78 In patients, the direct effect of
`tositumomab alone is not clear. It is administrated often
`as a predose before the isotope-labelled tositumomab. This
`pre-dose was shown to exert a tumour-reducing effect, but
`on the contrary slowed down the effect of tositumomab
`linked with Iodine-131.77 These results suggest the need
`for humanised B1-like antibodies for CDC-independent
`treatment of B-cell malignancies.
`
`H U m a n i s e d a n t i b o d y ( t y P e i i )
`
`Ga-101 (ro5072759).
`GA-101 is a humanised type II anti-CD20 MoAb. GA-101
`is generated by grafting CDR sequences of the B-ly1
`anti-CD20 MoAb on framework regions of fully human
`IgG1-kappa germline sequences. Different elbow hinge
`sequences in the variable region were optimised for optimal
`induction of apoptosis. In addition, the Fc region has been
`glycoengineered, which results in a 50-fold higher affinity to
`human FcγRIII receptors.79 In cynomolgus monkeys, GA101
`was shown to have a superior efficacy for B-cell depletion in
`the tissues as compared with rituximab.80 Currently ongoing
`
`phase I and II clinical studies will demonstrate the efficacy
`of GA-101 and its unique property to enhance ADCC and
`apoptosis of B cells. The first clinical data in a rituximab-
`pretreated patient population showed a favourable toxicity
`profile and an overall response rate of 58%.81
`
`
`d i s C U s s i o n
`
`Although CD20-targeted therapy with rituximab has
`greatly enhanced the outcome of patients with B-cell
`malignancies, resistance to rituximab is still a major
`problem, resulting in non-response and early relapse of
`disease ( figure 4). Second- and third-generation anti-CD20
`MoAbs have been developed to overcome resistance to
`rituximab. To assess the additional value of new antibodies,
`two approaches are recognised, i.e. to show superior
`efficacy if compared head-to-head with rituximab or to yield
`significant responses in rituximab-refractory NHL patients.
`Resistance is determined by a complex combination of the
`three mechanisms of action of rituximab (CDC, ADCC
`and apoptosis) and a patient-specific microenvironment
`of the lymphoma. B-cell depletion studies in monkeys
`and mice have also demonstrated that distinct subtypes
`of B cells in the lymph nodes exert different mechanisms
`of cell-specific resistance.58,59 Therefore, the combination
`of each patient and each lymphoma subtype may have its
`unique mechanism of resistance. Understanding all these
`factors that contribute to resistance may eventually lead to
`an individual-patient-based anti-CD20 therapy.
`
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