Boosting ADCC and CDC activity by Fc engineering and evaluation of antibody effector functions

Methods 65 (2014) 105–113

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Boosting ADCC and CDC activity by Fc engineering and evaluation of antibody effector functions Christian Kellner, Stefanie Derer, Thomas Valerius, Matthias Peipp ⇑ Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian-Albrechts-University Kiel, Germany

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Article history: Available online 9 July 2013 Keywords: ADCC CDC Antibody engineering Fc modification Antibody glycosylation Antibody therapy

a b s t r a c t In recent years, therapy with monoclonal antibodies has become standard of care in various clinical applications. Despite obvious clinical activity, not all patients respond and benefit from this generally well tolerated treatment option. Therefore, rational optimization of antibody therapy represents a major area of interest in translational research. Animal models and clinical data suggested important roles of Fc-mediated effector mechanisms such as antibody dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) in antibody therapy. These novel insights into the mechanisms of action mediated by monoclonal antibodies inspired the development of different engineering approaches to enhance/optimize antibodies’ effector functions. Fc-engineering approaches by altering the Fc-bound glycosylation profile or by exchanging amino acids in the protein backbone have been intensively studied. Here, advanced and emerging technologies in Fc-engineering resulting in altered ADCC and CDC activity are summarized and experimental strategies to evaluate antibodies’ effector functions are discussed. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Monoclonal antibodies represent established therapeutic options in the treatment of autoimmune diseases and cancer [1–4]. Depending on the special characteristics of the respective malignancy and the specific target structures, different effector mechanisms mediated by monoclonal antibodies may account for their therapeutic activity in vivo. In cancer therapy, Fc-mediated mechanisms such as ADCC and CDC have been suggested as being especially important for successful therapeutic intervention [3]. In various animal models and controlled clinical trials, the contributions of selected mechanisms of action have been partially unraveled for different therapeutic antibodies currently in clinical use. For example serum application in addition to rituximab infusion has been suggested to potentially increase rituximabs’ clinical efficacy in vivo [5,6] and C1q knockout in selected mouse models resulted in loss of therapeutic activity of rituximab [7,8], suggesting complement fixation to be important for tumor elimination. In other animal models, for unknown reasons, a role for complement to rituximabs’ therapeutic activity was not observed [9–11]. Complement deposition as well as the generation of anaphylatoxins such as C5a has also been demonstrated to potentiate

⇑ Corresponding author. Address: Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian-Albrechts-University Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany. Fax: +49 431 597 5803. E-mail address: [email protected] (M. Peipp). 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.06.036

effector cell-mediated killing, indicating that the complement system and Fc receptor-mediated killing are functionally linked in certain situations [12,13]. Interestingly, complement fixation by rituximab was even demonstrated to impair ADCC activity and therapeutic activity probably by interfering with Fc receptor (FcR) binding [14,15]. More detailed analyses of these unexpected findings suggested that C3b deposition induced by antibodycoated target cells impairs Fc/FccRIIIa interaction, thereby limiting NK activation and ADCC [14]. Collectively, available data may suggest that depending on tumor type, target antigen, tumor burden, tumor location and availability of components of the hosts/patients’ immune system only a selected repertoire of effector functions is available [16–18]. Consequently, potent complement fixation is beneficial when induction of CDC is possible, but in certain situations, it may prevent other effector mechanisms from being effective and thereby even compromise antibodies’ therapeutic effect. Many animal models suggested an important role for recruitment of effector cells via FcR engagement. While both, knockout of activating FcR and prevention of FcR signaling resulted in an almost complete loss of therapeutic activity mediated by rituximab or trastuzumab in mice, knockout of the inhibitory FccRIIb resulted in enhanced therapeutic activity [9,19]. These data were further underlined by analysis of isotype switch variants which show differential ratios of binding to activating and inhibitory FcR [10]. In these experiments, antibodies with a relative higher affinity to activating FcR than binding to the inhibitory FccRIIb demonstrated

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more potent therapeutic activity. In contrast, engagement of the inhibitory FccRIIb resulted in enhanced therapeutic activities of both DR5 antibodies triggering target cell apoptosis and agonistic CD40 antibodies revealing a complex role of different FcR in antibody therapy [20–23]. The importance of FcR engagement in antibody therapy was further supported by analyses of FcR polymorphisms in patients treated with monoclonal antibodies. In several studies, patients with homozygous expression of the FccRIIIa-V158 allele and/or the FccRIIa-H131 allele demonstrated higher response rates and prolonged overall survival [24,25]. FccRIIIa-V158 binds human IgG1-Fc with higher affinity than FccRIIIa-F158 resulting in enhanced ADCC activity [26]. The functional consequences of FccRIIa-H131 vs. FccRIIa-R131 engagement are less well defined (Fig. 1). Moreover, although intensively studied in mouse models, the relative contribution of different effector cell populations (i.e. NK cells, monocytes, macrophages, granulocytes) expressing different sets of activating and inhibitory FcR is still not fully clarified in the human system and may vary between different antibodies and disease entities [11,27,28]. Based on these observations, different antibody engineering approaches to potentially enhance antibody therapy became evident [29–31]. Here, different Fc engineering approaches are summarized and experimental strategies to evaluate engineered antibodies’ effector functions are discussed.

2. Modifying the protein backbone to enhance ADCC and CDC activity

identified an Fc variant (S239D–I332E) with enhanced FccRIIIa binding affinity that also showed stronger binding activity to FccRIIa and FccRIIb [34]. Similar to the triple variant described by Shields, antibodies harboring the S239D–I332E double amino acid substitutions triggered stronger NK cell-mediated ADCC. Interestingly, this variant was even more efficient in inducing NK cell-mediated ADCC than the triple Fc variant. In the background of rituximab the S239D–I332E variant triggered NK cell-mediated ADCC to a higher extent and showed a modest enhancement in macrophage-mediated antibody-dependent cell-mediated phagocytosis (ADCP), while CDC activity was not affected. Interestingly, this antibody variant achieved a 50% reduction in B-cell numbers in monkeys at an approximately 50-fold reduced dose level compared to its non-engineered counterpart (rituximab) [34]. This Fc variant was also tested in the background of a CD19 antibody [36,37]. While the non-engineered CD19 antibody did not show any B-cell depletion activity in monkeys, the Fc-optimized variant potently eliminated B cells in this model system [38]. Together, these data suggest that enhancing Fc affinity to FccRs by protein-engineering may be beneficial in cancer immunotherapy. To date no systematic, direct comparisons between the various protein-engineered Fc variants with different Fc receptor binding profiles have been reported. Since the relative contribution of the various effector cell populations expressing distinct FccR repertoires is not finally resolved, such data might be important to identify the most promising variants for further clinical development.

2.1. Protein-engineering to enhance ADCC activity

2.2. Protein-engineering to enhance CDC activity

Fc-engineering by exchanging critical amino acids in the protein backbone was a logical consequence to manipulate Fc/FcR interactions. In a pioneering approach, Shields and colleagues exchanged all solvent exposed amino acids in human IgG1-Fc to alanine, thereby identifying various variants with altered FcR binding profiles and effector functions [32]. By combining variants with single amino acid exchanges, a triple variant (S298A–E333A–K334A) was identified with increased FccRIIIa binding affinity, while binding to FccRIIa and FccRIIb was reduced. This modification increased ADCC activity with human NK cells, especially with donors homozygously expressing the FccRIIIa-F158 allele. Subsequently, a variety of Fc variants with enhanced FccRIIIa binding resulting in more potent ADCC activity were identified by using different approaches, such as rational engineering based on information from Fc/FcR co-crystal structures, yeast display and bacterial display [33–35]. Many of the described amino acid exchanges resulted in enhanced FccRIIIa binding but differed in their binding profile for the remaining FcR (Table 1, figure ure2). Lazar and colleagues

Similar to optimization of ADCC activity, engineering the protein backbone could be applied to achieve improvement of IgG1 antibodies’ capacities to trigger CDC. Based on findings that linked binding of the initial complement component C1q to the CH2 domain of IgG1 [39], several studies analyzed the impact of distinct amino acid substitutions in this region on complement activation [40,41]. Thereby, crucial amino acids have been identified for C1q ligation to the IgG1 CH2 domain. While Idusogie and colleagues found residues K326 and E333 to be involved in C1q binding, Moore and colleagues analyzed 38 antibody variants among which they unraveled residues S267, H268 and S324 to be crucial for effective CDC [40,41]. Interestingly, combination of the amino acid substitutions K326W (3-fold C1q binding) and E333S (1.5-fold C1q binding) resulted in higher C1q binding capacity (5-fold) but no further increase in CDC activity. Interestingly this variant almost completely lost its ADCC activity (Fig. 3; Table 2). However, loss of ADCC activity was prevented by substitution of lysine 326 and glutamic acid 333 by alanine residues, resulting in the

Fig. 1. FcR polymorphic sites associated with a favorable clinical response. The polymorphic sites that have been associated with favorable clinical responses in selected clinical trials are indicated in the background of co-crystal structures of Fc receptor and human IgG1-Fc. (A) FccRIIa/Fc co-crystal structure ([111]; pdb-file: 3RY6). (B) FccRIIIa/Fc co-crystal structure ([58]; pdb-file: 3SGJ). While the FccRIIIa-V/F158 polymorphism has been demonstrated to impact affinity of human IgG1-Fc binding and ADCC activity, a similar clear functional consequence has not been found for the FccRIIa-H/R131 interaction with human IgG1-Fc [112]. In contrast, a clear impact on human IgG2 binding has been demonstrated.

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C. Kellner et al. / Methods 65 (2014) 105–113 Table 1 Selected engineered IgG1-Fc variants with enhanced ADCC activity. Variant

FccRIIIa binding

FccRIIb binding

IIIa/IIb profile

ADCC induction

fold reduction in EC50 value

Complement activation

Wildtype S298A–E333A–K334A S239D–I332E S239D–I332E–A330L F243L–R292P–Y300L–V305I–P396L AME-D (specific mutations not disclosed)

" "" """ """" """ """

" ; "" """ "(") n.d.a

1 10 4 9 7 n.d.a.

" """ """ """ """ """

 10–100 10–100 10–100 10–100 5–10

+ n.d.a. +  n.d.a. n.d.a.

Reference

[32] [34] [34] [35] [33]

IIIa/IIb, fold FccRIIIa binding/FccRIIb binding; EC50, effective concentration 50%; ", enhanced activity/binding compared to wt.; ;, reduced activity/binding compared to wt.; n.d.a., no data available (Reprinted with permission [107]).

Fig. 2. Schematic representation of IgG1 variants with Fc modifications. The IgG model structure is based on the pdb-file [113] provided by Dr. Mike Clark (http:// www.path.cam.ac.uk/~mrc7/). Amino acid positions that are modified by Fc protein-engineering and fucosylation status are indicated. (A) wildtype IgG1, (B) afucosylated IgG1, (C) ADCC-optimized IgG1, (D) CDC-optimized IgG1, (E) IgG1-IgG3 mixed isotype 113F (see also Table 1/2). Yellow = fucose; green/yellow = carbohydrate structures.

Fig. 3. Proposed model of interaction between CDC-optimized Fc variant K326W–E333S and C1q. Fc variants with enhanced CDC activity display higher affinity C1q binding. The underlying mechanisms of high affinity C1q binding are not fully understood, since to date co-crystal structures of engineered Fc variants with C1q have not been reported. In the C1q–IgG1 assembly model proposed by Gaboriaud and colleagues the respective positions of amino acid exchanges in the CDC-optimized K326W–E333S Fc variant are highlighted. From this model it might be speculated that the indicated positions of amino acid exchanges result in altered higher affinity protein–protein interactions, although structural alterations cannot be excluded. (A) Crystal structure of IgG1 antibody b12 (pdb-file: 1HZH; [114]). (B) Space-filling representation of the proposed model of interaction between C1q subunit B and human IgG1 (assembly structure kindly provided by Dr. C. Gaboriaud and Dr. G.J. Arlaud; based on pdb-files: 1HZH and 1PK6 [115]).

double-mutant K326A–E333A – displaying 2.5-fold enhanced CDC activity and unaffected ADCC activity similar to wild type Fc. A 6.9-fold improvement in CDC activity was achieved by the combination of amino acid substitutions reported by Moore and colleagues (S267E-H268F-S324T). The single mutations also

enhanced CDC, although to a lesser extend: S267E (3.0-fold increased), H268F (2.0-fold increased) and S324T (1.9-fold increased). The CDC-optimizedtriple-mutant S267E-H268F-S324T showing high cytolytic potential, displayed substantially decreased ADCC activity. ADCC activity was restored by the insertion of two

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Table 2 Selected engineered IgG1-Fc variants with enhanced CDC activity. Variant Wildtype K326W K326W–E333S S267E–H268F–S324T S267E–H268F–S324T–G236A–I332E IgG1/IgG3 chimera

ID

EFT EFT + AE 113F

C1q binding (KD in nM)

C1q fold Binding

CDC (fold) potency

ADCC (fold) potency

Reference

48

1 3 5 47

1 2 2 6.9 23 3.7

1 0 0 0.045 1.2 1

[40] [40] [41] [41] [41,42]

1.0

7.1

n.d.a., no data available (Reprinted with permission [107]).

Table 3 Approaches to manipulate antibody fucosylation. Technique to manipulate fucosylation level

Fucosylation level

Reference

Unmodified/mutated cell line (YB02, LEC13) GNTIII overexpression Co-expression of heterologous GDP-6-deoxy-Dlyxo-4-hexulose reductase FUT8 gene knockout FUT8 siRNA knockdown Antibody expression in the presence of the glycosidase inhibitor kifunensine Zinc-finger nuclease mediated knockdown of GDP-fucose transporter gene

Variable Variable Variable

[44,46] [51] [108]

Afucosylated Afucosylated variable

[55] [109] [54]

Afucosylated

[110]

additional amino acid substitutions (G236A and I332E) (Table 2, Fig. 2), resulting in ADCC activity similar to wild type IgG1 and an even further enhancement in CDC activity (Table 2). Besides introducing individual amino acid substitutions to enhance CDC activity, also alternative approaches have been developed. For example, Natsume et al. designed mixed IgG1/IgG3 Fc variants with significantly enhanced CDC activity (Fig. 2; Table 2; see below) [42]. 3. Engineering the Fc-bound carbohydrate structure to enhance ADCC activity Early studies on structure/function relationships revealed that the presence of the complex type oligosaccharide attached to Asn297 in IgG1 antibodies was critical for FcR and complement binding. Lack of Fc glycosylation led to dramatic structural changes within the CH2 domain [43], resulting in a closed conformation almost incompatible with FcR and complement binding. The potential importance of Fc glycosylation for the cytotoxic activity of therapeutic antibodies was further supported by comparing antibody preparations of CAMPATH-1H from different expression cell lines [44]. These antibodies demonstrated distinct glycosylation profiles and significantly differed in their capacity to trigger ADCC. Consequently, controlled manipulation of the Fc-bound oligosaccharide to modulate Fc-mediated effector functions became one major area of interest in antibody engineering [45]. Especially variations in the content of fucose and sialic acid were correlated with altered ADCC activity [46,47]. Different strategies have been developed allowing rational alterations of the glycosylation profile to specifically improve antibodies’ capacity to trigger ADCC. 3.1. Reducing fucose level Reduction in core fucosylation to enhance FcR binding and ADCC activity could be achieved by different means. The most obvious way was to use cell lines displaying a reduced capacity to incorporate fucose in the antibody-attached oligosaccharide. Especially LEC13 cells, a CHO mutant, almost lack any fucosylation

activity [48]. This cell line is well suited for the production of antibodies with significantly reduced fucose levels at lab scale. Unfortunately this cell line was not very well suited for industrial scale production [49]. In another approach, overexpression of N-acetyl glucosaminyl transferase III (GnT-III) in production cell lines led to the attachment of a bisecting GlcNAc residue. The presence of this residue critically impacts multiple subsequent enzymatic glycosylation reactions in the Golgi complex. In that case, the bisected oligosaccharide cannot serve as a suitable substrate for a1,6-fucosyltransferase (a1,6-FucT) [50]. Using this strategy, an anti-neuroblastoma antibody was engineered by tetracycline-regulated expression of the GnT-III enzyme in CHO cells. The observed enhancement in ADCC activity (20-fold) correlated with the level of bisected, non-fucosylated oligosaccharides [51]. Similar observations were reported for an engineered variant of rituximab [52], and for a chimeric CD19 antibody produced in HEK-293 [53]. Subsequently, additional strategies have been pursued to reduce core fucosylation (Table 3) [51,52,54,55]. Especially knockout of the FUT8 gene encoding a1,6-FucT allowed the production of antibodies which were completely non-fucosylated [55]. From a mechanistic/structural point of view, different molecular mechanisms may account for the higher affinity FccRIIIa binding of non-fucosylated antibodies resulting in enhanced ADCC activity. Structural comparisons between fucosylated and nonfucosylated antibodies revealed that only subtle conformational changes in a limited region of IgG1 Fc were observed [56]. Ferrara and colleagues suggested that the high affinity binding of nonfucosylated Fc to FccRIIIa is mediated by interactions formed between the receptor carbohydrate attached to N-162 and regions of the Fc part that are only accessible when it is non-fucosylated [57]. More refined analysis of the interaction between non-fucosylated Fc and FccRIIIa by examination of co-crystal structures suggested that besides interactions between the Fc/FcR protein backbone, also the N-glycan structure attached at N-162 of FccRIIIa interacts with the Fc-bound N-glycans (Fig. 4) [58,59]. The carbohydrate-carbohydrate interface area covers about 12% of the total interface area and stabilizes the Fc/FcR complex. The presence of fucose inhibits this interaction by steric hindrance and negatively affects the dynamics of the receptor binding site by limiting the flexibility of Tyr-296 to adapt to an active conformation allowing formation of a high-affinity complex. Since FccRIIIa and FccRIIIb are the only human Fcc receptors which are glycosylated at this position, the proposed interactions between the FccRIIIa-attached carbohydrate and the Fc portion might explain the observed selective affinity increase for FccRIII [57–59]. The impact of the FccRIIIa-V/F158 gene polymorphism on ADCC mediated by non-fucosylated antibodies was investigated using variants of rituximab. Non-fucosylated rituximab more potently triggered ADCC than its fucosylated counterpart, irrespective of the FccRIIIa polymorphism [60]. The use of non-fucosylated rituximab reduced the difference in ADCC activity mediated by lowaffinity FccRIIIa-F158 and high-affinity FccRIIIa-V158 expressing effector cells. Therefore, it is speculated that non-fucosylated antibodies may display improved therapeutic activity for all patients

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Fig. 4. Proposed models in gaining affinity to FccRIIIa. Fc variants with enhanced NK cell-mediated ADCC activity display higher affinity FccRIIIa binding due to different proposed mechanisms. (A) Co-crystal structure of glycosylated FccRIIIa and human IgG1-Fc. Carbohydrate-carbohydrate interactions contribute to Fc/FcR interaction (B) Presence or absence of fucose residue modulates carbohydrate-carbohydrate interactions. (C) Protein engineering may modulate direct protein–protein interactions. By combined glyco- and protein-engineering, lack of fucose may further enhance Fc/FcR interaction. Illustrations were generated according to pdb-file 3SGJ [58].

independent of the FccRIIIa phenotype. While target cells opsonized with non-fucosylated antibodies were more effectively phagocytosed by neutrophils [61], in another study low-fucose content was correlated with impaired neutrophil mediated ADCC, suggesting that removal of fucose may not universally increase ADCC activity [62]. In animal models using human FccRIIIa transgenic mice, non-fucosylated antibodies demonstrated significantly improved therapeutic activity [63]. Non-fucosylated antibodies already have entered clinical trials [64–66] with Mogamulizumab (KW-0761) being approved in Japan for the treatment of relapsed or refractory adult T-cell leukemia–lymphoma. Whether nonfucosylated antibodies will indeed demonstrate superior clinical activity than their fully fucosylated counterparts is not easily proven, since in most cases no clinical data are available for the fully fucosylated counterpart (e.g. GA101, KW-0761) [67]. 3.2. Impact of galactose, mannose, bisecting GluNac and sialic acid While the correlation of fucose content and ADCC activity has been confirmed and clearly demonstrated in several antibody backgrounds, the impact of other sugar residues in the Fc-bound glycan structure on immune effector functions is less well defined. Differences in galactosylation were demonstrated not to affect ADCC activity [46]. The impact of a bisecting sugar has been analyzed elegantly by enzymatic remodeling of an antibody directed against CD20. In this model incorporation of a bisecting sugar residue resulted in 10-fold enhanced ADCC activity [68]. The impact of high mannose type glycans on ADCC activity is not fully resolved since high mannose content also correlated with reduced fucose content, thereby complicating data interpretation [47]. Higher levels of terminal sialic acids have been demonstrated to diminish NK cellmediated ADCC [69,70]. Interestingly, the mechanism by which sialic acids impact ADCC activity differed between antibody preparations. Data from Scallon and colleagues suggested that selected antibodies containing higher levels of sialic acid showed reduced Fc affinity to FccRIIIa, resulting in diminished ADCC activity [70]. Interestingly, in the background of other specificities differences in sialic acid content had no impact on FccRIIIa binding, but demonstrated reduced antigen binding capacity [70]. In summary,

modulation of sialylation in contrast to manipulation of fucose levels impacts antibodies’ ADCC activity by different means. To produce non-sialylated antibodies Naso and colleagues modified the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) to allow secretion in mammalian host cells lines [69]. Secreted expression of sialidase A in host cells expressing monoclonal antibodies quantitatively removed antibodies’ terminal sialic acids in the tissue culture supernatant. The purified molecules consistently triggered stronger ADCC than their sialylated counterparts [69]. 3.3. Glyco-engineering by antibody production in non-mammalian hosts or by carbohydrate remodeling Many glyco-engineering approaches focused on modifying mammalian host cell lines frequently used in antibody production (e.g. CHO), but also non-mammalian host cells such as insect, yeast and plant have been analyzed [71]. Especially yeast and plant cells have been extensively studied and genetically modified to allow production of antibodies with defined carbohydrate structures similar to molecules expressed in mammalian expression systems [72,73]. Since the numbers of clinically approved antibodies are growing these production systems may represent less expensive alternatives to mammalian expression systems that can be used to produce antibodies with a desired glycosylation profile [4]. Besides engineering the respective expression host used in antibody production, also glyco-engineering approaches have been proposed that make use of specific enzymes allowing rational remodeling of antibodies’ Fc-bound N-glycan structures. Although this strategy allows controlled attachment of well-defined carbohydrate structures, resulting in active molecules with optimized effector functions, this approach may generate additional costs by the more complex production process [68,74]. 4. Combined protein- and glyco-engineering While most Fc engineering approaches focused on modifications either in the protein backbone or in the Fc-attached carbohydrate structure, also combined approaches have been reported.

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Reducing the fucose level of an antibody harboring the ADCC-optimized triple variant S298A–E333A–K334A resulted in a further enhancement in NK cell-mediated ADCC activity [49]. Interestingly, when a similar approach was applied using variants with enhanced FccRIIIa binding affinity published by Lazar (S239D–I332E, S239D–I332E–A330L), a similar stronger ADCC activity was not observed although FccRIIIa binding was enhanced by approx. 10-fold [75,76]. These data suggested that protein-engineered variants and glyco-engineered variants gain FccRIIIa affinity by different means, e.g. stronger protein–protein interactions by amino acid substitutions and altered interactions between Fc- and FcR-bound carbohydrates (Fig. 4). In terms of cytotoxicity, these data suggest that for maximal NK cell-mediated ADCC triggered by FccRIIIa-engagement, a certain threshold has to be overcome, that is already reached by selected Fc variants or by glyco-engineering. To date these data have not been confirmed in a meaningful in vivo model, therefore it is not clear whether this finding holds true in a more complex in vivo situation. Natsume and colleagues analyzed the impact of reduced levels of fucose in the background of the 113F mixed isotype variant. As expected, no differences in CDC activity were observed, but interestingly lower levels of fucose were correlated with enhanced NK cell-mediated ADCC activity, further demonstrating that IgG protein-engineering approaches can be combined with glyco-engineering strategies [42]. Recently, aglycosylated Fc variants with restored binding activity to selected FccR by introducing additional amino acid exchanges have been published [77,78]. IgG1 variants with very unique FcR binding profiles and biologic functions were identified. In one approach introduction of amino acid substitutions in the glycosylation motif around amino acid position N297 led to aglycosylated Fc variants when produced in a mammalian host cell line. In the second approach, a novel bacterial-based screening system was used for identifying novel Fc variants. In the background of trastuzumab, the identified E382V–M428I variant expressed in Escherichia coli demonstrated selective binding to FccRI. No binding to other activating receptors such as FccRIIa and FccRIIIa or to the inhibitory FccRIIb receptor was observed. The glycosylated version of the trastuzumab E382V–M428I variant produced in a mammalian expression host, bound to all Fcc receptors similar to clinical grade trastuzumab. Importantly, E. coli-expressed trastuzumab harboring the E382V–M428I double mutation, but not the glycosylated trastuzumab E382V–M428I variant or clinical grade trastuzumab, was able to trigger killing of Her2-positive target cells with dendritic cells as effectors [78]. An explanation for these findings may be attributed to the differential engagement of the inhibitory FccRIIb receptor, while E. coli-expressed trastuzumab harboring the E382V–M428I double mutation selectively engages FccRI and lacks binding to FccRIIb, transtuzumab and the glycosylated trastuzumab E382V–M428I variant potently engage FccRIIb therby potentially attenuating the cytotoxic effector function. Together, these examples and additional recent reports [79] demonstrate that by combining protein- and glyco-engineering approaches, Fc domains can be engineered to display unique FccR and C1q binding and selectivity profiles, resulting in unique spectra of effector mechanisms.

5. Evaluation of effector functions The development of antibodies for clinical application, especially in the later phases of clinical testing is time consuming and expensive [1]. Relevant preclinical assay systems which in the ideal case reliably predict the clinical success rate already during the development process would dramatically reduce costs.

Antibody candidates in clinical development can be described by a variety of biochemical and biophysical parameters. Using sophisticated techniques such as high performance liquid chromatography (HPLC) and mass spectrometry (MS) even microvariations in amino acid composition and heterogeneity due to altered glycosylation can be assessed [80,81]. To analyze structure/function relationships additional biological assay systems are required that could not easily be standardized and have to be carefully designed to avoid potential pitfalls [82]. 5.1. Evaluation of effector functions in vitro Direct, solely Fab-mediated effector functions, such as ligand blockade, inhibition of proliferation or direct induction of apoptosis can be analyzed by high throughput screening in an automated fashion. For these assay systems well characterized tumor cell lines or transfectants with a defined genetic background displaying specific characteristics such as multi drug resistance or mutations which are also found in patients are available [83,84]. Many colorimetric or fluorescence-based assay systems (MTT, MTS, Alamar Blue etc.) are commercially available and can be applied [82]. By adding human serum or plasma also CDC activity of a set of antibodies can be tested in a similar straightforward and automated fashion [85,86]. These in vitro assays are well suited to compare panels of novel constructs to a ‘‘gold standard’’, for example a clinically approved antibody. To evaluate effector cell-mediated killing mechanisms such as ADCC, more complex assay conditions are required. Traditionally, ADCC is assessed in chromium or europium release assay systems, but also LDH release and flow cytometry-based assay system have been reported [87–89]. While chromium- and europium-based assays as well as flow cytometry-based assay systems allow strict discrimination between target and effector cell death, LDH release assays are more difficult to control, since it could not easily be discriminated whether LDH is released by dying effector cells or dying target cells or both. In addition, the LDH release assay is dependent on high levels of LDH in the target cells. This may not always be the case, resulting in decreased sensitivity of this assay system with selected target cells. In flow cytometry-based test systems the formation of target and/or effector cell aggregates may compromise data evaluation, especially when antibodies trigger homotypic aggregation [82]. Since primary tumor and effector cells could not easily be expanded or kept in culture, often immune effector cells freshly isolated from healthy volunteers in combination with well-characterized established cell lines or primary tumor cells are used [37]. As an alternative to freshly isolated immune effector cells, also genetically modified cell lines like NK-92 transduced to express human FccRIIIa have been used [90]. By applying these assay systems, the capacity of a given antibody to trigger selected effector cell populations can be assessed. Using unfractionated human whole blood or by adding isolated serum proteins to the assay system, the impact of high concentrations of immunoglobulin, complement factors or soluble Fcc receptors could be addressed [91–94]. In addition, by using effector cells or serum from genotyped donors, the impact of certain polymorphisms found in human effector molecules (e.g. FccR or complement proteins) can be analyzed [26]. Nevertheless, effector cells and plasma isolated from tumor patients may display killing characteristics different from the respective blood components derived from healthy donors. Therefore, data derived from these experiments have to be interpreted with care. Especially an allogeneic setting of target and effector cells may compromise data interpretation. For example, NK cell-mediated target cell lysis is more tightly inhibited in an autologous setting with matched target cell-expressed MHC class I molecules and respective effector cell-expressed KIR receptors. Similarly, also myeloid effector cells are regulated by various

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inhibitory surface receptors such as leukocyte Ig-like receptors (LIRs) that may affect FcR-mediated activation [68]. 5.2. In vivo evaluation of Fc-optimized antibodies In vivo models are considered as being essential in preclinical testing of therapeutic agents. For monoclonal antibodies or their engineered derivatives special considerations are required and in vivo data in xenografted models have to be interpreted with care. Antibodies have been analyzed in different animal models on a routine basis to determine in vivo body and/or plasma retention times and other basic pharmacokinetic parameters such as tissue distribution. Monoclonal antibodies targeting tumor cell-associated antigens were frequently tested in xenografted tumor models. Enhanced activity of antibody variants that were optimized to display stronger ADCC activity has been demonstrated in several mouse models using transgenic or non-transgenic animals. For example, superior anti-tumor activity of non-fucosylated trastuzumab has been demonstrated in human FccRIIIa transgenic mice [63]. Since antibodies engineered toward a higher affinity to human FccR receptors also often exert an enhanced affinity for murine FcR, the beneficial effects of Fc engineering can be demonstrated in non-transgenic animal models [36,95]. In addition, also tumor models using NOD/Shi-scid-IL-2Rgnull (NOG) mice have been used to test Fc-optimized antibodies. In these animals autologous human immune effector cells were engrafted [96]. Not many Fc-engineering approaches to enhance antibodies’ CDC activity have been systematically tested in mouse models so far. This may be due to limitations of the in vivo evaluation of CDC using human antibodies in xenograft mouse models. For example, complement-regulatory proteins expressed by human tumor cell lines do not perfectly match with murine complement components thereby making data interpretation on contribution to the efficacy of therapeutic antibodies difficult. Recently, Sato and colleagues used NOG mice to evaluate human complement functions in a small animal model by injecting human serum in combination with monoclonal antibodies. In this model, a rituximab-derived CDC-optimized antibody showed stronger anti-tumor effects than rituximab [97]. Mouse models are well suited in demonstrating antibodies’ activity in vivo, but the models may be affected by the genetic background and characteristics of the respective cell line and the mouse strain used for xenotransplantation experiments. As outlined, various fundamental aspects in antibody therapy have been addressed in animal models, but some concerns remain in translating findings to the human situation [9,27]. In terms of FcR-mediated effector functions the human and murine FcR system is not identical. Significant differences in tissue distribution of selected FcR exist [98]. In addition when human antibodies are tested in non-humanized mouse models Fc selectivity for murine FcR is a critical parameter to consider [99]. This situation may be even more complex when Fc-optimized antibodies are evaluated, since the FcR binding profile may differ between the murine and human system. Especially when the relative contribution of various effector cell populations to the in vivo activity is analyzed, results have to be interpreted with care. For example, the murine FccRIV is expressed on phagocytic effector cell populations (monocytes, granulocytes) but is absent on NK cells. The human homologue, FccRIIIa, is exquisitely expressed on NK cells and macrophages but in contrast to the murine system no expression is found on granulocytes [98]. In addition for the FccRIIIb found to be expressed on human granulocytes no murine homologue exists. A further complexity in data interpretation derives from differences in the interactions of human antibodies with human and mouse FcRn, resulting in differences in pharmacokinetic profiles, further

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complicating direct translation of efficacy results into the human context. These limitations may in part be overcome by humanizing mouse models by transgenic expression of selected human Fc receptors, xenografting human effecor cells or by engrafting immunodeficient mice with human hematopoietic stem cells to establish a humanized immune system displaying a more complex mixture of various human immune effector populations [96,100,101]. Besides careful interpretation of anti-tumor activity, other critical points to consider are toxic side effects that may be underestimated since antibodies intended for application in humans often do not sufficiently cross-react with the murine homologue. Along the same lines, toxic side-effects observed in mice may be due to cross-reactivity, not necessarily observed in the human situation. Carefully taking these potential limitations into account, mouse models are invaluable to gain first proof of concept if a novel or modified antibody has activity in vivo and how it performs in comparison to a ‘‘gold standard’’, e.g. a clinically approved monoclonal antibody. Non-human primate models have been used to investigate pharmacokinetic properties and to monitor toxicity mediated by novel antibodies. However, similar to mouse models cross-reactivity with primate antigens and with primate Fc receptors are crucial factors that have to be carefully addressed. Differences in antibody affinity for the primate homologue and a potentially different expression profile of the targeted antigen may underestimate or overestimate toxic side effects [102]. Nevertheless, as a surrogate model for therapeutic activity, potency in depletion of selected leukocyte populations, such as B cells has been used to compare the activity of wildtype and Fc-engineered antibodies [34,38,103]. For example, an Fc-optimized CD19 antibody engineered by exchanging two amino acids in the Fc domain demonstrated potent B-cell depletion activity, while its non-engineered counterpart was unable to trigger any depleting activity [62]. In addition, different Fc-engineered variants of rituximab showed more efficient B-cell depletion activity in cynomolgus monkeys [34,42]. Although studies in non-human primates may most closely resemble the human situation, these models cannot substitute carefully designed clinical phase I studies. Even after careful preclinical testing in different rodent and non-human primate models, side effects of novel antibodies may not show up. Probably one of the most dramatic examples of this fact was the so-called ‘‘Tegenero-incident’’. The agonistic CD28 antibody TGN1412 did not show cytokine release in primates, but triggered deleterious cytokine storms in six healthy volunteers [104]. Importantly, studies designed to unravel the cause of failure in predicting these severe side effects in preclinical models suggested that a suitable in vitro test system with human cells was more predictive than in vivo testing in cynomolgus monkeys [105,106]. Thus, differences in tissue distribution and different biological functions of homologous target antigens in human and non-human primates as well as differences in antibody affinity and potency in eliciting effector mechanisms, may severely compromise data interpretation.

6. Conclusions Today, monoclonal antibodies are frequently applied in various clinical situations. Unfortunately, not all patients benefit from antibody therapy. A more detailed understanding of the mechanisms of action of therapeutically applied monoclonal antibodies has inspired the development of next generation antibodies which can be rationally designed to trigger distinct effector functions. As outlined above, Fc engineering approaches represent powerful technologies in boosting ADCC and CDC activity. Together with a more profound in depth understanding in the development and progression of diseases, the described approaches will hopefully

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allow the design of fit-for-purpose monoclonal antibodies with customized effector mechanisms that are optimally suited for application in various clinical settings. Acknowledgements Professor Dr. Christine Gaboriaud and Professor Dr. Gerard J. Arlaud are kindly acknowledged for providing the pdb coordinate files used to illustrate the putative IgG1/C1q interactions of CDCoptimized Fc variants. This work was supported by research grant 2007.065.2 from the Wilhelm Sander Stiftung (Neustadt, Germany) and a research grantDJCLS D 12/19 from the Deutsche José Carreras Leukämie Stiftung e.v. (Munich, Germany). We apologize to all investigators whose important work in the field could not be cited because of space limitations. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

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