review
`mAbs 5:1, 22–33; January/February 2013; © 2013 Landes Bioscience
`
`Epitope interactions of monoclonal antibodies
`targeting CD20 and their relationship to
`functional properties
`
`Christian Klein,1,* Alfred Lammens,2 wolfgang Schäfer,4 Guy Georges,4 Manfred Schwaiger,3 ekkehard Mössner,1
`Karl-Peter Hopfner,2 Pablo Umaña1 and Gerhard Niederfellner3
`
`1Discovery Oncology; Pharma research and early Development (preD); roche Glycart AG; Schlieren, Switzerland; 2Department of Chemistry and Biochemistry; Gene Center;
`Ludwig-Maximilians University Munich; Munich, Germany; 3Discovery Oncology; Pharma research and early Development (preD); roche Diagnostics GmbH; Penzberg,
`Germany; 4Large Molecule research; Pharma research and early Development (preD); roche Diagnostics GmbH; Penzberg, Germany
`
`Keywords: Rituximab, obinutuzumab, ofatumumab, GA101, structure, type I, type II, non-Hodgkin lymphoma, immunotherapy,
`leukemia
`
`Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; CDC,
`complement-dependent cytotoxicity; CDR, complementarity-determining region; CLL, chronic lymphocytic leukemia; DLBCL,
`diffuse large B cell lymphoma; FcγR, Fcγ receptor; FL, follicular lymphoma; Ig, immunoglobulin; NHL, non-Hodgkin
`lymphoma; MS, multiple sclerosis
`
`Several novel anti-CD20 monoclonal antibodies are currently
`in development with the aim of improving the treatment
`of B cell malignancies. Mutagenesis and epitope mapping
`studies have revealed differences between the CD20 epitopes
`recognized by these antibodies. recently, X-ray crystallography
`studies confirmed that the Type i CD20 antibody rituximab
`and the Type
`ii CD20 antibody obinutuzumab (GA101)
`differ fundamentally in their interaction with CD20 despite
`recognizing a partially overlapping epitope on CD20. The
`Type i CD20 antibodies rituximab and ofatumumab are known
`to bind to different epitopes. The differences suggest that
`the biological properties of these antibodies are not solely
`determined by their core epitope sequences, but also depend
`on other factors, such as the elbow hinge angle, the orientation
`of the bound antibody and differential effects mediated by the
`Fc region of the antibody. Taken together, these factors may
`explain differences in the preclinical properties and clinical
`efficacy of anti-CD20 antibodies.
`
`Introduction
`
`CD20 is a transmembrane cellular protein that has been vali-
`dated as a therapeutic target for treatment of B cell malignan-
`cies1 (Fig. 1A). CD20 is highly expressed by over 95% of B cell
`lymphocytes throughout their development, from the pre-B
`cell stage until their final differentiation into plasma cells, but
`is absent on the hematopoietic stem cell.2 Moreover, CD20 is
`believed to exist predominantly as a tetramer on the cell surface.
`It is also largely believed to be not usually shed or internalized
`
`*Correspondence to: Christian Klein; Email: christian.klein.ck1@roche.com
`Submitted: 09/30/12; Revised: 10/31/12; Accepted: 11/03/12
`http://dx.doi.org/10.4161/mabs.22771
`
`upon antibody binding, meaning that therapeutic antibodies
`may be expected to recruit immune effectors cells and mediate
`sustained immunologic activity.3 The physiological function of
`CD20 remains unclear,1 although evidence suggested that it may
`be involved in calcium signaling downstream of B cell antigen
`receptor activation.4
`Rituximab
`(MabThera®; Rituxan®, Roche/Genentech/
`Biogen IDEC) was the first monoclonal antibody to be approved
`for the treatment of lymphoma, and it has changed the treat-
`ment of non-Hodgkin lymphoma (NHL) and chronic lym-
`phocytic leukemia (CLL),5 particularly in combination with
`chemotherapy where it has been shown to improve survival
`compared with chemotherapy alone.6-10 More recently, the use
`of rituximab in maintenance therapy has been shown to fur-
`ther improve outcomes in patients with follicular lymphoma
`(FL).11-16 This has established rituximab’s position as a standard-
`of-care therapy in the treatment of NHL and CLL.17-19 Other
`anti-CD20 antibodies have been introduced into use, including
`ofatumumab (Arzerra®; Genmab/GlaxoSmithKline), which is
`a human antibody approved for refractory CLL,20,21 and tositu-
`momab (Bexxar®, GlaxoSmithKline) and ibritumomab tiuxetan
`(Zevalin®, Spectrum), which are murine antibodies used clini-
`cally as radioimmunoconjugates.22 Ongoing research aims to
`develop novel anti-CD20 antibodies with improved properties
`and greater clinical efficacy. Critical to this process is a better
`understanding of the mechanisms by which anti-CD20 anti-
`bodies act and the relative contributions of different modes of
`action to clinical efficacy.
`After binding to CD20-positive cells, antibodies are
`thought to trigger at least three different effector functions:
`(programmed) cell death (also termed as direct cell death or
`apoptosis), antibody-dependent cellular cytotoxicity (ADCC)
`or phagocytosis (ADCP) and complement-dependent cyto-
`toxicity (CDC).3,23 Anti-CD20 antibodies are categorized as
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`Type I or Type II according to their mode
`of CD20 binding and their primary mecha-
`nism for killing CD20-positive cells24-29
`(Table 1).
`This review article will focus on the applica-
`tion of anti-CD20 monoclonal antibodies to B
`cell malignancies; however, it should be noted
`that some of the antibodies discussed in this
`review have also been approved30 or are being
`investigated31 in the treatment of non-cancer
`indications (e.g., multiple sclerosis, rheuma-
`toid arthritis, systemic lupus erythematosus).
`
`Type I and Type II CD20 Antibodies
`and their Effector Functions
`
`Most existing anti-CD20 antibodies, includ-
`ing
`rituximab, veltuzumab, ocrelizumab
`and ofatumumab, are categorized as Type I
`(Table 2). These antibodies are character-
`ized by their ability to induce a transloca-
`tion of CD20 into large lipid microdomains
`or ‘lipid rafts’ within the plasma membrane
`upon binding.26,32,33 This clustering process
`enhances the recruitment and activation of
`complement, and hence Type I antibodies
`exert potent CDC.25,26 However, the contribu-
`tion of complement activation to the depletion
`of B cells in vivo remains unclear.3,34 Another
`characteristic feature of Type I antibodies is
`that B cells can be bound by twice as many
`Type I antibodies compared with Type II anti-
`bodies,27,35 most likely due to different binding
`geometries. The biological significance of this
`is unknown, but it has been hypothesized that
`the 2:1 stochiometry could be explained by
`Type I antibodies binding between two CD20
`tetramers, thereby crosslinking tetramers with
`two antibodies bound per tetramer, whereas
`Type II antibodies may bind within a tetra-
`mer, resulting in only one antibody bound per
`CD20 tetramer29,36 (Fig. 2). In line with this,
`the two known Type II anti-CD20 antibod-
`ies tositumomab (or B1) and obinutuzumab
`(GA101) (Table 2), do not induce accumu-
`lation of CD20 upon antibody binding in
`insoluble lipid rafts and show relatively little
`CDC activity.25,27 On the other hand, Type II
`antibodies are more potent than Type I anti-
`bodies in inducing homotypic adhesion and
`direct cell death.24,25,27 Although this form of
`cell death was initially described as apoptosis,
`recent studies have demonstrated that it is a
`non-apoptotic form of direct cell death that
`follows an actin-dependent enhancement of
`cell-to-cell contact, the rupturing of lysosomes
`
`Figure 1. (A) The structure and topology of CD20 and the epitopes recognized by rituximab,
`ofatumumab and GA101. (B) Sequence alignment of CD20 epitopes recognized by CD20
`antibodies based on published information. Core epitope residues are boxed in light blue.
`For 2F2 (ofatumumab), core epitope assignment is based on published work from Teeling
`et al. 46. For residues labeled in blue experimental evidence suggests a role in 2F2 binding.
`For the other antibodies, the following coloring scheme has been applied based on Pepscan
`results and FACS binding data of amino acid exchange mutants: green, almost any exchange
`tolerated at this position; brown, non-conservative exchange tested and not tolerated at this
`position; orange, conservative exchange tested and tolerated at this position; red, also con-
`servative exchanges not tolerated at this position; black, position has not yet been evaluated.
`italic font indicates that Pepscan and FACS binding results are discordant. Since the FACS
`binding results better reflect the native protein context, the coloring in such instants was
`based on the FACS binding data.
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`within the cytoplasm28,37,38 and the generation of reactive oxygen
`species, but does not show the classical hallmarks of apoptosis
`such as DNA laddering or caspase dependence.39
`The ADCC and ADCP activity of anti-CD20 antibodies is
`mediated by the interaction of their Fc region with FcγRIIIa and
`is not affected by the Type I or Type II character of the antibody.
`FcγRIIIa is expressed on various immune effector cells, most
`prominently macrophages/monocytes and natural killer cells.
`FcγRIIIa crosslinking by binding to CD20 on target cells stimu-
`lates release of lytic enzymes by the effector cells and induces cell
`killing or promotes the phagocytosis of the target CD20 positive
`cell.3 Two variants of FcγRIIIa have been identified in humans: a
`predominant lower affinity form with a phenylalanine at position
`158 (FcγRIIIa-158F) and a higher affinity form with valine at
`this position (FcγRIIIa-158V).40-42 The binding of the Fc region
`of antibodies to FcγRIIIa is dependent on interactions between
`the carbohydrate moieties of both the FcγRIIIa and antibody.43
`Notably, ADCC activity does not differ between Type I and
`Type II anti-CD20 antibodies,3 but antibodies such as GA101
`have been engineered for enhanced affinity for FcγRIIIa leading
`to an increased ability to bind and recruit effector cells and hence
`a higher ADCC level.27,44 The contribution of ADCC to the clin-
`ical activity of antibodies remains to be established. However,
`the expression of the higher affinity FcγRIIIa-158V genotype
`in lymphoma patients has been shown to be associated with an
`improved response to rituxumab (mono-) therapy,40,45 suggesting
`that enhanced FcγRIIIa affinity may confer a clinical advantage.
`Recently, Beers and colleagues46 demonstrated an increased
`potency in depleting B cells from human CD20 transgenic
`mice of Type II antibodies compared with Type I antibodies.
`They attributed much of this disparity to the Type I antibody-
`mediated internalization of CD20 by B cells leading to reduced
`recruitment of macrophages (ADCP) and degradation of CD20/
`antibody complexes. The authors also noted that the type of
`disease affected the degree of internalization, with most cases
`of CLL and mantle cell lymphoma showing rapid CD20 inter-
`nalization; this was in contrast to FL and DLBCL cells, which
`were more resistant to CD20 loss. The internalization process
`was promoted by the inhibitory FcγRIIb on target B cells and
`investigations have suggested that rituximab can crosslink CD20
`and FcγRIIb on the same cell (in cis), whereas Type II antibodies
`do not appear to have this function47 (Fig. 3).
`Anti-CD20 antibodies possess complementarity-determining
`regions (CDR) that bind to a specific epitope on the antigen.
`Mutational analyses and peptide scanning studies have revealed
`differences between antibodies in their CD20 epitopes.29,48,49
`Recently, three-dimensional crystallographic representations of
`several antibodies in complex with CD20 confirmed fundamen-
`tal differences in their interactions with CD20 (Fig. 4) [ritux-
`imab,50 C2H7 (ocrelizumab),51 ofatumumab,52 GA101].
`Structurally, CD20 comprises four hydrophobic membrane-
`spanning domains, two extracellular loops (one of approximately
`44 amino acids and a smaller one of approximately seven amino
`acids), and intracellular N- and C-terminal regions (Fig. 1A).
`The intracellular regions of CD20 can undergo phosphorylation
`upon antibody binding, thereby mediating cellular signaling.1
`
`Table 1. Characteristics of Type i and ii antibodies
`Type I antibodies
`Type II antibodies
`Class i epitope
`Class ii epitope
`Localize CD20 to lipid rafts
`Do not localize CD20 to lipid rafts
`High CDC
`Low CDC
`ADCC activity
`ADCC activity
`Full binding capacity
`Half binding capacity
`weak homotypic aggregation
`Homotypic aggregation
`Cell death induction
`Stronger cell death induction
`
`rituximab, ocrelizumab (2H7),
`ofatumumab (2F2)
`
`GA101, tositumomab (B1)
`
`ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-
`dependent cytotoxicity; mAb, monoclonal antibody.
`
`Most of the epitopes involved in antibody recognition are located
`within the larger extracellular loop. Recently, Niederfellner and
`colleagues29 mapped the epitopes recognized by anti-CD20 anti-
`bodies. They showed that, despite recognizing an overlapping
`epitope on the large extracellular loop of CD20, Type II anti-
`bodies bind in a different orientation than Type I antibodies.
`For example, the core epitope of GA101 (a Type II antibody) is
`formed by residues 172–178, whereas the Type I antibody ritux-
`imab targets the more N-terminally comprising residues 168–
`175, with 170–173 contributing most essentially. For binding of
`Type II antibodies, asparagine 176 (N176) is a critical residue
`(Fig. 1B), whereas this residue does not seem to make any con-
`tacts with CD20-bound Type I antibodies, as exemplified by the
`crystal structure of rituximab (Fig. 5). The crystal structure of
`the GA101–CD20 epitope peptide complex confirmed that the
`shift in the core epitope resulted in a fundamentally different
`orientation of GA101 with respect to CD20. Based upon the cur-
`rently available data, we have generated a model of rituximab and
`GA101 bound to CD20 (Fig. 6). Ofatumumab, another Type I
`antibody, binds to both the large and small CD20 extracellular
`loops,48,52 as discussed below.
`
`Type I CD20 Antibodies
`
`Rituximab. Rituximab is a Type I chimeric (human–mouse)
`immunoglobulin (Ig)G1 anti-CD20 antibody. The CD20 epit-
`ope recognized by rituximab and other mouse-derived antibod-
`ies spans amino acid residues 168–175 of the CD20 protein,
`with the ANPS motif at residues 170–173 on the large extracel-
`lular loop appearing to be of critical importance29,33,48,50,53 (Fig.
`1B). These key residues have been shown to form a network of
`hydrogen bonds with residues of the surrounding CDR loops.51
`The particular importance of the alanine residue at position 170
`(A170) and the proline residue at position 172 (P172) was shown
`by site-directed mutagenesis studies taking advantage of the
`fact that rituximab binds only human, but not mouse, CD20.
`Introducing the 170ANP172 motif into mouse CD20 conferred
`binding of rituximab. The importance of the 170ANPS173 region
`for rituximab binding in humans has also been established by the
`screening of libraries of phage-displayed peptides with different
`
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`Table 2. Characteristics of selected anti-CD20 monoclonal antibodies
`
`Names
`
`Development status
`(indication)
`
`Description
`
`Type I or II
`
`Epitope
`
`rituximab
`
`Approved (NHL, DLBCL,
`CLL)
`Phase 3 (MCL, DLBCL)
`
`Chimeric igG1
`
`Ofatumumab (2F2;
`HuMax-CD20)
`
`Approved (CLL)
`Phase 2 (DLBCL)
`
`Human igG1
`
`veltuzumab (iMMU-
`06; hA20)
`
`Phase 2 (NHL)
`
`Humanized igG1κ
`
`Ocaratuzumab (AMe-
`D, AMe-133)
`
`Phase 2 (NHL)
`
`Ocrelizumab
`
`Phase 3 (MS)
`
`PrO131921 (rhuMAb
`v114)
`
`Discontinued
`
`TrU-015
`
`Discontinued
`
`Humanized igG1 with
`Fab/Fc engineered
`to improve CD20 and
`Fcγriiia affinity
`
`Humanized igG1
`(2H7-based)
`
`Humanized igG1
`(2H7-based) Fc engi-
`neered to improve
`Fcγriiia affinity
`
`Single-chain CD20-
`targeting protein
`derived from 2H7 and
`with a human igG1
`hinge
`
`ibritumomab tiux-
`etan (Zevalin)
`
`Approved (FL)
`
`Murine igG1κ
`
`Tositumomab
`(Bexxar)
`
`Approved
`Orphan status in FL
`
`Murine igG2aλ
`
`Obinutuzumab
`GA101
`
`Phase 3 (DLBCL, NHL, CLL,
`refractory)
`
`Humanized igG1κ
`
`i
`
`i
`
`i
`
`i
`
`i
`
`i
`
`i
`
`i
`
`ii
`
`ii
`
`hOUBM3/6
`
`Preclinical
`
`Humanized igG1κ
`
`Unclear
`
`Large extracellular loop
`• Core epitope: 170ANPS173 region33
`• 182YCYSi186: contributes to conformational stability49
`• WPXWLE: functional significance unclear53
`• Contact region: positions 165–18248
`
`Large extracellular loop
`• Core epitope: FLKMESLNFIRAHT region48
`• T159K, N163D and N166D residues critical, mostly
`likely for conformational stability48
`Small extracellular loop
`• A74T, I76A and Y77S residues76
`
`Largely identical to rituximab (above)84
`
`Largely identical to rituximab (above)85
`
`Large extracellular loop
`• Core epitope: 170ANPS173 51
`• P168 and P170 contribute to binding51
`• Contact region: positions 165–18048
`
`Same as 2H7/ocrelizumab51
`
`Same as 2H7/ocrelizumab51
`
`Same as rituximab (above)61
`
`Large extracellular loop
`• Core epitope: 170ANPS173 33
`• Contact region: positions 170–18248
`
`Large extracellular loop
`• Core epitope: 172–176 region29
`
`Large extracellular loop
`• ES, RAHT and INIYN75
`• Not 170A or P172 75
`
`CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B cell lymphoma; FL, follicular lymphoma; ig, immunoglobulin; MCL, mantle cell lymphoma;
`NHL, non-Hodgkin’s lymphoma
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`Figure 2. Hypothetical model for the 2:1 binding ratio of Type i and Type ii CD20 antibodies
`binding to CD20 (tetramers, depicted in red). An explanation to explain the 2:1 binding stoi-
`chiometry between Type i and Type ii CD20 antibodies is to assume that (A) Type i antibodies
`bind between CD20 tetramer (inter-tetramer, depicted in red) resulting in accumulation in lipid
`rafts together with Fcγriib (gray oval). in contrast Type ii (B) antibodies may bind within one
`tetramer (intra-tetramer).
`
`sequences53 where P172 was found to have
`a particular importance, since rituximab
`binds the human ANPS sequence but not
`the corresponding murine SNSS sequence.53
`Furthermore, mutation of the alanine and
`proline at positions 170 and 172 in human
`CD20 to serine was shown to abolish ritux-
`imab binding.33,48 Asparagine 171 (N171)
`was also found to be a key residue for ritux-
`imab binding as any amino acid replacement
`at this position, except histidine, resulted in
`a substantial loss of binding affinity to pep-
`tides representing the extracellular CD20
`loop.29
`sug-
`also
`screening
`Phage-peptide
`gested that a second region of the epitope,
`182YCYSI186, contributes to the binding of
`rituximab through conformational stabi-
`lization.49 Furthermore, when Perosa and
`colleagues screened phage-display peptide
`libraries containing a repertoire of sequences
`of random 7- or 12-amino acid peptides they
`found that, while cyclic peptides mimick-
`ing the CD20 epitope were dependent on
`the 170ANPS173 motif, linear mimics that
`also bound rituximab required a differ-
`ent motif—WPxWLE—that does not cor-
`respond to any sequence present in CD20
`itself.53,54 While the WPxWLE motif appears to share
`some rituximab contact points with 170ANPS173, these
`regions are conformationally different and have been
`proposed as distinct epitopes.54 However, the func-
`tional role and significance of the WPxWLE sequence
`is unclear.
`Mutagenesis studies can identify residues affecting
`antibody binding, but cannot define the contact sites
`between the CD20 epitope and the antibody. The
`structure of the rituximab:epitope complex has been
`determined by co-crystallizing a synthetic peptide
`mimic of the extracellular loop epitope of CD20 (resi-
`dues 163–187) in complex with the antigen-binding
`fragment of rituximab.50 The bound CD20 peptide
`forms a cyclic conformation owing to a disulfide bond
`between two cysteine residues, C167 and C183. This
`structure comprises a short N-terminal coil (residues
`167–171), a 310 helix (residues 172–174), a small loop
`(residues 175–177) and a short C-terminal α-helix
`(residues 178 –184). The key 170ANPS173 motif is
`embedded in a cyclic, four-region pocket formed by
`the CDRs of the rituximab antibody (Fig. 5). Residues of the
`170ANPS173 motif bind to CDR residues via numerous hydrogen
`bonds and van der Waals contacts. In accordance with evidence
`that P172 has a critical role in antibody binding, this residue is
`deeply buried in the CD20/Ab interface and forms additional
`hydrophobic and hydrophilic contacts with residues at the bottom
`of the CDR pocket that are likely to be important in maintaining
`
`Figure 3. Hypothetical model for CD20 binding of Type i and Type ii CD20 antibod-
`ies explaining the impact of Fcγriib on internalization. (A) Type i antibodies such as
`rituximab may bind to CD20 in a conformation that allows simultaneous binding to
`Fcγriib and subsequent signaling followed by internalization in lipid rafts. (B) Type ii
`antibodies such as GA101 may bind in a conformation that does not allow simultane-
`ous binding to Fcγriib, thus resulting in reduced internalization.
`
`the conformational stability of the epitope-antibody complex.50
`The 182YCYSI186 region at the C-terminus of the large extracel-
`lular loop of CD20 also appears to play a role in rituximab bind-
`ing,49 most likely through the formation of the disulfide bond
`that induces the cyclic conformation of the epitope50 loop nec-
`essary for the binding of CD20 to rituximab.55 Abrogating the
`internal disulfide bridge (C167-C183) of the large extracellular
`
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`with a Fab region engineered to improve CD20-binding
`affinity. AME-133v has a ca. 13- to 20-fold greater bind-
`ing affinity for CD20 than rituximab.60 The Fc region
`has been modified to improve affinity for FcγRIIIa-158F
`and -158V genotypes. As a result, AME-133v shows
`greater in vitro activation of natural killer cells and 5- to
`7-fold more potent ADCC than rituximab.44,60 AME-
`133v recognizes the same epitope as rituximab.
`Ibritumomab. Ibritumomab, a murine IgG1κ Type
`I antibody, is the antibody from which rituximab was
`derived and hence targets the same epitope as rituximab.61
`A radiolabeled form of the antibody, 90Y-ibritumomab
`tiuxetan (Zevalin, Spectrum), is used in the treatment of
`indolent NHL62-64 and as consolidation therapy follow-
`ing induction.65,66
`Ocrelizumab. Ocrelizumab (PRO70769, Roche/
`Genentech) is a humanized anti-CD20 IgG1 Type I
`antibody that has been evaluated in a Phase 1/2 study
`in patients with relapsed/refractory FL and is currently
`in development for the treatment of multiple sclero-
`sis.67 Compared with rituximab, ocrelizumab shows
`lower CDC activity but greater ADCC activity and
`enhanced binding to the low-affinity FcγRIIIa vari-
`ant.67 Ocrelizumab is based on the murine Type I IgG2b
`antibody 2H7. The CDR loops of 2H7 are structurally
`similar to those of rituximab. Among the four CDR loops that
`interact with CD20, only one (H3) differs substantially from the
`rituximab counterpart in terms of residue sequence and confor-
`mation.51 2H7 was first thought to recognize exactly the same
`epitope as rituximab. Early studies confirmed that residues A170
`and P172 of CD20 are necessary for 2H7 binding, but suggested
`that they are not sufficient alone. Rather, the 162INxxN166 motif
`also appeared to be necessary for full binding of 2H7 in the pres-
`ence of A170/P172, possibly because these residues may stabilize
`the conformation of the 2H7:CD20 complex. Mutation of the
`QTSK motif present in murine CD20 to 156RAHT159 (as present
`in human CD20) also improved the binding of 2H7, but was not
`necessary for full binding. In addition, 2H7 appears to only bind
`the oligomeric form of CD20 (e.g., tetramers).33 Subsequent pep-
`tide scanning studies demonstrated that the core contact regions
`for 2H7 (CD20 positions 165–180) and rituximab (CD20 posi-
`tions 165–182) are almost identical.48 Crystallography has con-
`firmed that the CDR loops of 2H7, like those of rituximab, form
`a deep pocket enclosing the critical 170ANPS173 epitope motif of
`CD20.51 The P168 and P170 residues of 2H7 also form hydro-
`gen bonds with CD20, while P175, which occurs in both 2H7
`and rituximab, forms a hydrophilic interaction with CD20 that
`is oriented differently in the 2H7-CD20 and rituximab-CD20
`complexes. As with rituximab, the cyclic conformation of the
`2H7-CD20 complex is maintained by the disulfide bond of the
`peptide. The different structure of the H3 loop of 2H7, as com-
`pared with rituximab, alters the topology of the complex. These
`differences result in fewer binding interactions for 2H7, and
`hence a lower binding affinity, compared with rituximab.51
`PRO131921. PRO131921 (rhuMAb v114, Genentech) is a
`humanized IgG1 anti-CD20 antibody that was studied in two
`
`Figure 4. Published crystal structures of CD20 antibodies. (A) rituximab-CD20
`complex,48 (B) ofatumumab (no co-crystal structure is available),50 (C) 2H7-CD20
`complex,49 and (D) GA101-CD20 complex.29 The heavy chain is colored in darker
`shades, the peptides derived from CD20 are colored in red where appropriate.
`
`loop seems to completely destabilize the CD20 protein, since
`expression of a CD20 variant with a C167S exchange is barely
`detectable by western blot analysis after transient transfection of
`HEK293 cells.29
`The knowledge of the CD20 epitope was used to design ritux-
`imab variants in which point mutations were inserted into the
`CDR to improve the binding characteristics of the antibody.56
`Rituximab variants that bound to CD20 with enhanced avid-
`ity, or with a reduced off-rate, did not show improved activity in
`terms of CDC, complement fixation or rafting. However, a vari-
`ant with three mutational changes (H57DE/H102YK/L93NR/)
`was shown to mediate enhanced avidity-dependent ADCC and
`cell death.56
`In principle, genetic mutations in the rituximab epitope
`could reduce the binding and efficacy of the antibody, but clini-
`cal data in patients with DLBCL suggest that epitope mutations
`are very rare (0.4% of 264 patients at diagnosis and one of 15
`patients at relapse) and are not an important cause of failure
`of treatment with rituximab in combination with conventional
`chemotherapy.57
`Veltuzumab.
`hA20,
`(IMMU-106;
`Veltuzumab
`Immunomodics, Nycomed) is a humanized IgG1κ Type I anti-
`body in Phase 2 development for treatment of relapsed or refrac-
`tory NHL and autoimmune diseases58 (Table 2). Veltuzumab has
`CDRs largely identical to those of rituximab with the exception
`of one residue, suggesting that it binds to the same epitope.59
`Veltuzumab competes for CD20 binding with rituximab and
`shows similar specificity, avidity and in vitro activity.58,59
`AME-133v. AME-133v
`(Ocaratuzumab, LY2469298,
`MENTRIK) is a humanized IgG1 Type I antibody in Phase 2
`development. AME-133v is an optimized version of rituximab
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`Figure 5. Comparison of (A) rituximab (Type i) and (B) GA101 (Type ii) crystal structures in complex with CD20 peptide.29 while for rituximab N171 is
`deeply immersed and N176 has no contacts with the rituximab CDrs, N171 is not deeply immersed in the the GA101 CDrs and vice versa N176 makes
`contacts to residues F52/D57/D59 of GA101 supporting the C-terminal shift of the GA101 epitope.
`
`Phase 1 clinical trials, one for CLL and one for NHL.
`PRO131921 is derived from 2H7, but carries a modi-
`fied Fc region with enhanced affinity for FcγRIIIa.68
`PRO131921 interacts with the same epitope as ocreli-
`zumab.69 Clinical development has been discontinued.70
`TRU-015. TRU-015 is a single-chain CD20-targeting
`protein that was derived from 2H7 and has a human IgG1
`hinge that binds to the same epitope of 2H7.51 TRU-015
`was described to show reduced CDC activity but more in
`vitro and in vivo properties compared with rituximab.71
`Clinical development was discontinued.
`Ofatumumab. Ofatumumab is a human IgG1 Type
`I antibody that is approved for the treatment of patients
`with CLL refractory to fludarabine and alemtuzumab.20,21
`Ofatumumab is being studied in patients with lym-
`phomas either as a single agent or in combination with
`chemotherapy.58,72-74
`Like rituximab, ofatumumab shows Type I anti-
`CD20 activity, including CD20 rafting and CDC activ-
`ity,35,75 but binding studies suggest that ofatumumab
`recognizes an epitope different from that of rituximab.
`While the binding of rituximab is prevented by muta-
`tion of the A170/P172 residues, site-directed mutagenesis
`has shown that such mutations in the large extracellular
`loop of CD20 do not affect the binding of ofatumumab.
`Rather, the replacement of asparagine at position 163 (N163)
`or 166 (N166) with aspartic acid reduced ofatumumab binding
`by 50–75%. A triple mutant with mutations T159K, N163D
`and N166D did not bind ofatumumab at all.48,76 None of these
`single mutations affected rituximab binding, although the triple
`mutant showed slightly decreased binding. Peptide scanning
`analyses confirmed that ofatumumab (together with the four
`other human IgG1 or IgGM antibodies tested) does not recog-
`nize the A170/P172 motif. Instead, these human antibodies rec-
`ognize a particular region in the large extracellular loop (146FLK
`MES LNF IRA HTP160) that is N-terminal to A170 and P172
`(Figs. 1B and 4B). This region does not include the N163 and
`N166 residues shown by mutagenesis studies to be necessary for
`
`Figure 6. Three-dimensional models of (A) rituximab and (B) GA101. GA101
`binds to the same binding epitope region of CD20 as rituximab, but in a dif-
`ferent binding orientation. The molecular models were created by combining
`known structural data with the current knowledge and general understanding
`of antibody structure and membrane protein topology. The CD20 membrane
`protein model was created by combining the structural fragments of the crystal-
`lized CD20 antibody binding epitope and the transmembrane part of the Her2
`receptor as a typical example of a membrane spanning molecule with known
`3D information, and CD20 topology information.
`
`ofatumumab binding, suggesting that these residues indirectly
`contribute to the stability of the epitope rather than forming part
`of the binding site itself.48
`Peptide scanning and mutagenesis studies have revealed that
`the small extracellular loop of CD20 also contributes to the bind-
`ing of ofatumumab. Binding of ofatumumab was almost com-
`pletely prevented by the replacement of the entire small loop with
`an alternative sequence or by the insertion of three mutations
`(A74T, I76A and Y77S) in the loop. Neither the loop replace-
`ment nor these mutations affected the binding of rituximab.76
`These data confirm that ofatumumab recognizes an epitope
`distinct from that of rituximab, which comprises discontinuous
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`sequences across both the large and small extracellular loops of
`CD20 (Fig. 1A).
`According to crystallography, the region of the ofatumumab
`molecule that binds with CD20 comprises six CDR loops,
`which form a deep pocket. Around the periphery of the pocket
`are hydrophobic residues (Y32, W94, W53, I58, Y60, Y102 and
`Y105) and at the bottom of the pocket is a positively charged resi-
`due (R91).52 It should be noted that the crystal structure of the
`Fab fragment of ofatumumab was determined in the absence of
`CD2052 (Fig. 4B). The hydrophobic pocket formed by the CDRs
`of ofatumumab is thought to interact with hydrophobic residues
`on both the large and small extracellular loops of CD20, and
`possibly with the cell membrane itself. The negatively charged
`N-terminal E150 residue of the large extracellular loop of CD20
`is thought to interact with the positively charged R91 residue at
`the bottom of the CDR pocket of ofatumumab.
`The binding of ofatumumab to the large and small extracel-
`lular loop of CD20 was hypothesized to position ofatumumab
`closer to the surface of the CD20 cell membrane than antibod-
`ies binding the large loop. This could be expected to facilitate
`the deposition of activated complement on the cell surface and
`hence the amplification of the complement response.77 However,
`the impact of this is unclear as the CD20 extracellular loop is
`very small compared with the size of an antibody so that the
`antibody-