mAbs
`
`ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: https://www.tandfonline.com/loi/kmab20
`
`Evolution of anti- CD20 monoclonal antibody
`therapeutics in oncology
`
`Ezogelin Oflazoglu & Laurent P. Audoly
`
`To cite this article: Ezogelin Oflazoglu & Laurent P. Audoly (2010) Evolution of anti- CD20
`monoclonal antibody therapeutics in oncology, mAbs, 2:1, 14-19, DOI: 10.4161/mabs.2.1.10789
`To link to this article: https://doi.org/10.4161/mabs.2.1.10789
`
`© 2010 Landes Bioscience
`
`Published online: 01 Jan 2010.
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`mAbs 2:1, 14-19; January/February 2010; © 2010 Landes Bioscience
`
`Evolution of anti-CD20 monoclonal antibody
`therapeutics in oncology
`
`ezogelin Oflazoglu* and Laurent P. Audoly
`
`Merck Bioventures; Discovery Biologics; Merck & Co., inc.; rahway, NJ USA
`
`Key words: CD20, NHL, CLL, monoclonal antibody, next generation anti-CD20 antibodies, ADCC, CDC, ADCP, PCD,
`rituximab
`
`Approval of an anti-CD20 chimeric monoclonal antibody,
`rituximab, has
`revolutionized
`cancer
`treatment and
`also validated CD20 targeting for providing benefit and
`improvement of overall response rate in B cell malignancies.
`Although many patients have benefited from the treatment of
`rituximab, there are still significant numbers of patients who
`are refractory or develop resistance to the treatment. Here we
`discuss pre-clinically well-defined potential mechanisms of
`action for rituximab and review the ways next generation anti-
`CD20 monoclonal antibodies can potentially exploit them to
`further enhance the treatment of B cell malignancies. Although
`the relative importance of each of these mechanism remains
`to be established in the clinic, well-designed clinical trials will
`help to define the efficacy and understanding of which effector
`activity of modified next generation anti-CD20 mAb will be
`important in the treatment of B-cell malignancies.
`
`Introduction
`
`The treatment of B cell malignancies has undergone substantial
`change since initial marketing approval in 1997 of the chimeric
`anti-CD20 antibody rituximab for the treatment of both aggres-
`sive and indolent subtypes of Non-Hodgkin lymphoma (NHL).1
`Rituximab is approved for use as monotherapy and in combi-
`nation with chemotherapeutics. Treatment with rituximab has
`resulted in significant improvement in overall response rates and
`survival of patients with NHL.2-9 Despite these improvements,
`there are significant numbers of relapsed/refractory lymphoma
`patients1,10 and infusion related adverse events in the clinical
`setting.11
`Several studies have suggested that rituximab activity is
`dependent on CD20 expression12 for both direct killing activity
`via CD20 signaling e.g., programmed cell death (PCD), sensi-
`tization of cells to chemotherapy13 and engagement of effector
`pathways,13 i.e., complement dependent cytotoxicity (CDC),
`antibody dependent cellular cytotoxicity (ADCC) and antibody
`dependent cellular phagocytosis (ADCP) (Fig. 1).13 Furthermore,
`
`*Correspondence to: Ezogelin Oflazoglu; Email: Ezogelin_oflazoglu@merck.
`com
`Submitted: 11/24/09; Accepted: 11/30/09
`Previously published online:
`www.landesbioscience.com/journals/mabs/article/10789
`
`passive immunization has been hypothesized as another potential
`mechanism for improving efficacy of rituximab, which supported
`the idea of using rituximab in a maintenance setting.14 In this
`study, it was shown that rituximab induced apoptosis of lym-
`phoma cells promotes phagocytosis by dendritic cells and cross-
`priming of CD8 positive cytotoxic T lymphocytes. At this stage,
`whether this immunization effect is specific to rituximab or to
`chemotherapeutic regimens is still unclear in the clinical setting.
`
`Programmed Cell Death Activity
`
`Rituximab can induce PCD as a result of CD20 signaling and
`this activity can be augmented when rituximab is hypercross-
`linked via a secondary antibody or binding via Fc gamma recep-
`tors in vitro.15 Although how this crosslinking activity is achieved
`in vivo still remains to be proven, primary tumors derived from
`rituximab treated chronic lymphocytic leukemia (CLL) patients
`were shown to express activated caspase-3 and caspase-9 indicat-
`ing the presence of PCD activity in vivo.16 A xenograft model
`has also shown that increased expression of anti-apoptotic Bcl-2
`family proteins can result in rituximab insensitivity.17 Whether,
`a similar phenomenon applies to primary tumors remains to
`be determined. Recently, Lim et al.13 have summarized studies
`where they compared the ability of rituximab to deplete human
`CD20 transgenic mouse B cells in vivo in the presence or absence
`of a second transgene encoding high levels of Bcl-2, which blocks
`the intrinsic apoptosis pathway.13 They reported that B cells
`expressing the Bcl-2 transgene were relatively resistant to apop-
`totic stimuli in vitro whereas in vivo they were just as susceptible
`to rituximab activity as B-cells lacking the transgene.13 The con-
`clusion from these studies was that in a fully syngeneic system,
`induction of the intrinsic apoptosis pathway is not important
`for subsequent B cell depletion.13 While all these studies suggest
`that rituximab is involved in promoting cell death, whether this
`mechanism is critical for the depletion of CD20 positive target
`cells in vivo remains to be determined.
`
`Fc-Fc Gamma Receptor Interaction Dependent
`Activity
`
`Fc binding to Fc gamma receptors expressed on monocytes, mac-
`rophages, natural killer (NK) cells and neutrophils can lead not
`only to ADCC and ADCP activities but also direct killing via
`
`14
`
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`Figure 1. Mechanism of action of rituximab. rituximab can induce cell death via several mechanisms. Antigen-antibody (Ag-Ab) complexes formation
`and Fc-Fc gamma receptor (Fcγr) complexes binding to CD20 can induce programmed cell death (PCD) by triggering the intrinsic pathway of apop-
`totic caspase activation via the Bcl-2 family proteins (Signal A) and mitochondrial outer membrane permeabilisation (MOMP) (Signal B). in antibody-
`dependent cell-mediated-cytotoxicity (ADCC), rituximab recruits effector cells by binding to their Fcγ receptors and this triggers effector cells to
`release of pre-forming proteins and proteases thus resulting in target cell death. in antibody-dependent cellular-phagocytosis (ADCP) rituximab
`recruits monocytes/macrophages by binding to their Fcγ receptors and this results in engulfment of antibody coated tumor cells. in complement-
`mediated cytotoxicity (CDC), rituximab activates complement cascade and generates membrane attack complexes and as a result induce cell death.
`MOr, mechanisms of resistance; sCD20, soluble CD20; Cir, complement inhibitory receptors.
`
`CD20 signaling due to hypercrosslinking.15-18 The early preclini-
`cal evidence for the involvement Fc-Fc gamma receptor interaction
`came from an in vivo study with the xenograft model, showing
`that rituximab activity is dependent on the gamma chain asso-
`ciated activating Fc receptors.19 Additional supporting evidence
`comes from a clinical study showing a better response with ritux-
`imab in NHL patients with higher affinity allelic variants of Fc
`gamma IIIa receptor.20-23 However, this correlation has not been
`observed in CLL patients,24 and it is hypothesized that this might
`be due in part to lower level of CD20 expression and the presence
`of higher levels of soluble CD20 in plasma.12 It also has been
`noted that in mice monocytes/macrophages are the main effector
`cells that contributes to the activity of rituximab compared to
`the NK cells and neutrophils in humans.25-27 Moreover, maximal
`monoclonal antibody (mAb) response activity has been shown to
`be dependent on intact compartments of the reticulo-endothelial
`system, as shown in experiments that surgical limitation of the
`
`hepatic blood supply correlated with lower B cell depletion.25-27 It
`has also been demonstrated that mouse Fc gamma receptor IV, a
`homolog of human Fc gamma receptor IIIa, is strongly involved
`in the effects of human IgG1.28 It is expressed on murine mono-
`cytes and macrophages, but not on murine NK cells, whereas
`human Fc gamma receptor IIIa is expressed on human NK cells,
`neutrophils and monocyte/macrophages.28 Due to differences
`of Fc gamma receptor expression profiles in human and mouse
`NK cells, the impact of NK cell activity in vivo preclinical mod-
`els might not be relevant in, and translate to, clinical settings.
`Although human NK cells are shown to be mediating ADCC
`activity in vitro,29 questions remain as to whether NK cells are
`the key cells in mediating rituximab activity or whether, ADCC
`is the dominant mechanism in tissues.
`
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`mAbs
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`Table 1. Development status and mechanism of action of monoclonal antibodies targeting CD20 that are approved or in clinical development
`
`mAb
`
`Format
`
`Indication
`
`Manufacturer
`
`Binding site
`
`Comments
`
`Phase Dev
`
`cigG1
`
`NHL, rA
`
`Genentech, Biogen
`
`Type i
`
`PCD, ADCC,
`CDC, ADCP
`
`Approved in US
`1997
`
`rituximab
`rituxan®
`MabThera®
`
`reditux
`
`Y90-ibritumomab tiuxetan
`Zevalin®
`
`i131tositumomab
`Bexxar®
`
`Ofatumumab
`Arzerra®
`
`Dr. reddy Laboratories
`
`Same as rituximab
`
`Biosimilar
`
`Biogen iDeC
`
`Same as rituximab
`
`Low ADCC
`
`NHL
`
`NHL
`
`NHL
`
`cigG1
`
`migG1
`
`migG2a
`
`higG1
`
`GlaxoSmithKline
`
`CLL, NHL,
`rA
`
`Genmab, GlaxoSmithKline
`
`Different than
`rituximab Type ii
`
`Different than
`rituximab
`
`Approved in india
`2007
`
`Approved in US
`2002
`
`Approved in US
`2003
`
`Approved in US
`2009
`
`Phase 3
`
`Phase 2
`
`Phase 2
`
`Low CDC
`
`Low Koff
`High CDC
`
`High ADCC
`Low CDC
`Low Koff
`High CDC
`
`High PCD
`High ADCC
`Low CDC
`
`Ocrelizumab
`
`higG1
`
`NHL, rA
`
`Genentech, roche, Biogen
`
`Same as rituximab
`
`veltuzumab
`
`higG1
`
`NHL, iTP
`
`immunomedics
`
`Same as rituximab
`
`Obinutuzumab GA101
`
`higG1
`
`CLL, NHL
`
`Glycart roche
`
`Type ii
`
`AMe-133v
`
`TrU-015
`
`PrO131921
`(version 114)
`
`higG1
`
`SMiP
`
`NHL
`
`rA
`
`Applied Molecular
`evolution, eli Lilly
`
`Trubion Pharma, wyeth
`
`higG1
`
`CLL, NHL
`
`Genentech
`
`LFB-r603/eMAB-6
`
`cigG1
`
`CLL
`
`GTC Biotherapeutics, LFB
`Biotechnologies
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`High ADCC
`
`Phase 2
`
`High ADCC
`Low CDC
`
`High CDC
`High ADCC
`
`Phase 2
`
`Phase 1/2
`
`High ADCC
`
`Phase 1
`
`N/A, information is not available; NHL, non-Hodgkin lymphoma; rA, rheumatoid arthritis; CLL, chronic lymphocytic leukemia; iTP, idiopathic thrombo-
`cytopenic purpura; PCD, programmed cell death; ADCC, antibody dependent cellular cytotoxicity; ADCP, antibody dependent cellular phagocytosis;
`SMiP, small modular immunopharmaceutical drug composed of human igG1 Fc and hinge regions (hinge, CH2 and CH3) linked directly to an
`anti-CD20 scFv.
`
`Complement Dependent Cytotoxicity Activity
`
`Although Fc-Fc gamma receptor interaction is widely accepted
`to be critical for the activity of rituximab in vivo, CDC activi-
`ties are still being debated.18,28 Supporting evidence for the role
`of CDC comes from studies where rituximab was shown to be
`capable of C1q binding and inducing CDC against malignant B
`cells in vitro.30-32 Moreover, when rituximab activity was tested
`in complement deficient or cobra venom inactivated comple-
`ment studies rituximab was shown to exhibit reduced activity
`in vivo.33,34 Additional evidence comes from studies in patients
`showing that soon after rituximab infusion, complements were
`being consumed in vivo, and that addition of complement ex
`vivo was capable of restoring the activity of rituximab in CDC
`assays.35,36 Although earlier studies show the critical role of CDC
`activity of rituximab, it should be noted that this preclinical
`activity of rituximab was not sufficient to deplete B cells in vivo
`nor it did correlate with the expression of complement inhibitory
`receptors.25,37,38 Yet it has also been shown that deposition of C3b
`not only facilitated the removal of rituximab:CD20 complexes
`from the B lymphoma cells by Fc gamma receptor expressing
`
`macrophages through the process of trogocytosis, but it also
`blocked the interaction between the Fc domain of rituximab and
`Fc gamma receptor IIIA on NK cells thereby decreasing ADCC
`activity.35,39-41 Human genetic polymorphism correlation provides
`an additional line of evidence suggesting the involvement of com-
`plement in the mechanism of action of rituximab. A study inves-
`tigating the impact of C1qA polymorphisms on the efficacy of
`rituximab demonstrated that follicular lymphoma patients with
`a low C1q expressing A allele correlated with enhanced rituximab
`responses compared to those patients with the high C1q express-
`ing G allele.42 All these studies indicate that CDC plays a role in
`rituximab activity, but whether this activity is critical and has a
`positive or negative impact remains unresolved.
`
`Next Generation Anti-CD20 Monoclonal Antibodies
`
`While successes, limitations and elucidation of the mechanism of
`action of rituximab have increased our understanding and helped
`advance the engineering of next generation anti-CD20 mAbs
`with the goal of improving the efficacy and decreasing associated
`adverse events, one still needs to better understand the potential
`
`16
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`interplay between the multiple proposed mechanisms of action-
`binding kinetics to different epitopes, PCD, CDC, ADCC and
`ADCP (Table 1). Depending on the proposed modification, next
`generation antibodies may be grouped in one of two categories:
`second or third generation anti-CD20 mAb.
`Second generation antibodies can be tailored to be human-
`ized or fully human with unmodified Fc domain, with the aim
`of reducing immunogenicity. Likewise, third generation anti-
`bodies can be modified to include engineered Fc domains with
`the aim of improving the therapeutic activity in all patients,
`particularly in genetically defined subpopulations that express a
`low affinity version of the Fc receptor on their immune effector
`cells. Second generation antibodies include ofatumumab, ocre-
`lizumab and veltuzumab. Compared to the other CD20 mAbs,
`fully human IgG1 ofatumumab is at the most advanced stage: it
`is approved in the US and undergoing regulatory review in the
`EU for the treatment of CLL.43-45 Ofatumumab binds a differ-
`ent CD20 epitope compared to rituximab and has a slower off
`rate such that over 3 hours the disassociation for ofatumumab is
`only 20–30%, but 70–80% for rituximab.46,47 Moreover, ofatu-
`mumab exhibits not only ∼10 fold higher CDC activity in ritux-
`imab sensitive tumor cell lines but also exhibits CDC activity
`in rituximab resistant cell lines.43,48 ADCC activities of ofatu-
`mumab have been shown to be similar to rituximab although
`it is a weaker PCD inducer than rituximab.43,48 Ocrelizumab is
`currently being developed for non-oncology indications, and has
`been shown to bind to the same CD20 epitope as rituximab,47,49
`but the molecule has relatively higher ADCC and low CDC
`activities compared to rituximab.50,51 Veltuzumab is a humanized
`IgG1 and has similar mechanisms as rituximab, with the excep-
`tion of a 2.5 fold slower off rate and higher CDC activity (mean
`EC50 of <100 ng/ml) compared to rituximab (EC50 of ∼150 ng/
`ml).52,53 Phase 1/2 studies with recurrent B cell lymphomas have
`shown a 53% overall response rate, including 6 patients with
`complete response at a median follow up of 12 weeks.54 Phase 2
`clinical trials using the new subcutaneous formulation of veltu-
`zumab for NHL, CLL and ITP patients are on going.55 Clinical
`trials testing veltuzumab and ofatumumab may provide further
`insights into the importance of different epitope on PCD activ-
`ity in the clinical context.
`Third generation anti-CD20 mAbs in early phases of clini-
`cal development include AME133v, Pro13192 (v114), GA101
`and R603/EMAB-6. TRU-015, is another third generation anti-
`CD20 molecule that is a small modular immunopharmaceutical
`drug composed of human IgG1 Fc and CH2 and CH3 hinge
`regions linked directly to an anti-CD20 scFv.56-58 It is slightly
`smaller than an IgG and has high ADCC and low complement
`activating ability. It is currently in Phase 2 clinical development
`for RA.59-62 AME-133v, an Fc protein engineered antibody, is cur-
`rently being evaluated in a Phase 1/2 dose escalation study using
`weekly intravenous doses for four consecutive weeks in patients
`with relapsed/refractory follicular B cell NHL. In vitro models
`have shown that the Fc domain of AME-133v binds to the low-
`affinity variant of Fc gamma RIIIa (FF or FV) with a higher
`affinity (mean EC50 <10 ng/ml) thereby improving killing of B
`cells ∼10 fold over rituximab.63,64 The improvement in efficacy
`
`of AME-133v that has been seen in preclinical settings has yet
`to be demonstrated in a clinical setting. Although the current
`clinical trial with AME-133v started in July of 2006 and had an
`estimated primary completion date of December 2008, clinical
`data remain unavailable as of October 2009.
`Pro131921 (v114), is another Fc protein engineered antibody
`and displays 30-fold greater binding to the low-affinity variant
`of Fc gamma RIIIa (FF or FV) than rituximab.65 In vitro, this
`binding affinity exhibits improved ADCC activity up to 10 fold
`more than rituximab. Preclinical studies in non-human primates
`showed that treatment with Pro13192 (v114) resulted in a dose-
`dependent reversible neutropenia and thrombocytopenia.65 Phase
`1/2 clinical studies to assess safety of escalating doses of Pro13192
`(v114) in patients with NHL and CLL were recently terminated.
`GA101 is a third-generation anti-CD20 mAb with a glyco-
`engineered Fc portion which exhibits improved binding affin-
`ity to FcgammaRIII by 50-fold, that results in a 10- to 100-fold
`increase in ADCC against CD20 positive NHL cell lines.66-70
`On the other hand, CDC activity of GA101 is much lower than
`rituximab in vitro.66-70 Moreover, GA101 depleted normal B
`cells as well as B cell lymphoma, significantly more than other
`CD20 directed antibodies, including rituximab Fc variants with
`improved ADCC activity.69,70 In vivo xenograft experiments in
`which cobra venom factor was used to inhibit complement activ-
`ity demonstrated that GA101 may be efficacious in that setting.71
`Structurally, GA101 contains a modified Fc domain hinge region
`that results in stronger induction of apoptosis of several NHL cell
`lines and primary malignant B cells.66 These modifications may
`provide GA101 with an increased therapeutic efficacy, leading
`to complete responses and long-term survival in xenograft mod-
`els of diffuse large B cell lymphoma and mantle cell lymphoma
`(MCL).71-74 In cynomolgus monkeys, GA101 compared to ritux-
`imab induced complete, rapid and long-lasting B cell deple-
`tion not only in peripheral blood but also in spleen and lymph
`nodes.74,75 It is still unknown whether this superior activity comes
`from PCD or decreased CDC activity compared to rituximab.
`LFB-R603/EMAB-6 is a chimeric third generation IgG1 and
`is produced in rat cell line YB2/0 using EMABLING technology
`thus resulting in naturally low fucose content in its Fc region.76
`Compared to rituximab, LFB-R603/EMAB-6 has similar CDC
`and PCD activities whereas FcγRIIIA binding and FcγRIIIA-
`dependent effector functions are higher and results in producing an
`ADCC plateau around 35% at 50 ng/ml, while rituximab induced
`less than 5% ADCC at the same concentration.76 Furthermore,
`LFB-R603/EMAB-6 induces higher ADCC activity against CLL
`cells than rituximab even when target cells express fewer CD20
`molecules.76 Although the improvement in efficacy of LFB-R603/
`EMAB-6 that has been seen in preclinical settings, the clinical
`activity is yet to be demonstrated in the future.
`
`Perspective
`
`In the face of the excitement and perhaps uncertainty generated
`by the next generation of anti-CD20 mAbs, their potential suc-
`cess in the treatment of B cell malignancies will depend on their
`clinically demonstrated safety and efficacy profiles. There is the
`
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`potential to achieve far better therapeutic efficacy than rituximab,
`and, significantly, demonstrate efficacy in rituximab-resistant
`populations. Additionally, all third generation antibodies appear
`to be Fc engineered (protein or glyco-engineering) with the hopes
`of improving the affinity of Fc-Fc gamma receptor interaction,
`thereby improving the ADCC activity. Although improvement
`of these interactions has increased ADCC activity in vitro, the
`significance of this mechanism is yet to be determined in the
`clinic. Furthermore, one should note that modulating Fc interac-
`tions can also affect other mechanisms of cell cytotoxicity such as
`ADCP activity and CD20 mediated apoptosis due to hypercross-
`linking via Fc gamma receptor.
`Although the mechanisms of action of each CD20 mAb are
`well-studied in preclinical settings, the variability seen in clinical
`response to rituximab may also depend on level of CD20 expres-
`sion, levels of circulating soluble CD20, presence and abundance
`of effector cells, CD20 binding epitope and kinetics, tissue dis-
`tribution and tumor burden. The predictive value of preclinical
`models in terms of quantification of the dose-concentration-effect
`
`relationship of rituximab using pharmacokinetic-pharmacody-
`namic analysis, identification of the individual factors influencing
`the response, relative importance of each of these mechanisms of
`action and resistance remains to be better understood and proven
`in the clinic. Well-designed clinical trials will help define and
`refine efficacy and provide increased understanding of which activ-
`ity of modified next generation anti-CD20 mAb will prevail. It
`is interesting to note that rituximab in combination with metho-
`trexate is also approved for treatment of adult RA patients who
`have had inadequate response to TNF antagonist therapies, and
`the product is also currently being studied in additional indica-
`tions. Evaluations of modified next generation anti-CD20 mAb as
`treatments for non-oncology indications can potentially add to our
`understanding of the critical activities required for efficacy.
`
`Acknowledgements
`We would like to thank Michele Mccolgan and Greg Roland for
`their help with the figure and the table, respectively.
`
`2.
`
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