Engineered antibody Fc variants with enhancedeffector functionGreg A. Lazar*†, Wei Dang*, Sher Karki*, Omid Vafa*, Judy S. Peng, Linus Hyun, Cheryl Chan, Helen S. Chung,Araz Eivazi, Sean C. Yoder, Jost Vielmetter, David F. Carmichael, Robert J. Hayes, and Bassil I. Dahiyat

Xencor, Inc., 111 West Lemon Avenue, Monrovia, CA 91016

Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved January 19, 2006 (received for review September 16, 2005)

Antibody-dependent cell-mediated cytotoxicity, a key effectorfunction for the clinical efficacy of monoclonal antibodies, ismediated primarily through a set of closely related Fc� receptorswith both activating and inhibitory activities. By using computa-tional design algorithms and high-throughput screening, we haveengineered a series of Fc variants with optimized Fc� receptoraffinity and specificity. The designed variants display >2 orders ofmagnitude enhancement of in vitro effector function, enableefficacy against cells expressing low levels of target antigen, andresult in increased cytotoxicity in an in vivo preclinical model. Ourengineered Fc regions offer a means for improving the nextgeneration of therapeutic antibodies and have the potential tobroaden the diversity of antigens that can be targeted for anti-body-based tumor therapy.

antibody-dependent cell-mediated cytotoxicity � Fc�R � proteinengineering � cancer

Monoclonal antibodies (mAbs) have enormous potential asanticancer therapeutics, with inherent advantages such as

specificity for target, low toxicity relative to small-molecule drugs,long half-life in serum, and the capacity for multiple cytotoxicmechanisms of action. There are eight approved anticancer anti-body (Ab) products and numerous more in development. Despitesuch widespread use, however, the potency of Abs as anticanceragents remains suboptimal. Patient tumor response data show thateven the most successful approved drugs provide incrementalimprovements in therapeutic success over single-agent chemother-apeutics. Many other promising Ab drugs, despite targeting anti-gens with favorable differential expression profiles, have failed inclinical trials because of insufficient demonstrable efficacy.

A promising means for enhancing the antitumor potency of Absis through enhancement of their ability to mediate cellular cytotoxiceffector functions such as Ab-dependent cell-mediated cytotoxicity(ADCC) and Ab-dependent cell-mediated phagocytosis (ADCP)(1). For the IgG class of Abs, ADCC and ADCP are governed byengagement of the Fc region with a family of receptors referred toas the Fc� receptors (Fc�Rs) (2). In humans, this protein familycomprises Fc�RI (CD64); Fc�RII (CD32), including isoformsFc�RIIa, Fc�RIIb, and Fc�RIIc; and Fc�RIII (CD16), includingisoforms Fc�RIIIa and Fc�RIIIb (3, 4). Fc�Rs are expressed on avariety of immune cells, and formation of the Fc�Fc�R complexrecruits these cells to sites of bound antigen, typically resulting insignaling and subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocy-tosis, and cytotoxic attack. All Fc�Rs bind the same region on IgGFc, yet with differing high (Fc�RI) and low (Fc�RII and Fc�RIII)affinities (5, 6). Furthermore, whereas Fc�RI, Fc�RIIa�c, andFc�RIIIa are activating receptors characterized by an intracellularimmunoreceptor tyrosine-based activation motif (ITAM), Fc�RIIbhas an inhibition motif (ITIM) and is therefore inhibitory.

Three critical sets of data support the role of Fc�R-mediatedeffector functions in Ab cancer therapy and the relationship be-tween Fc�Fc�R affinity and cytotoxic potency. First, xenograftstudies in Fc�R knockout mice indicate that activation receptorsare necessary and inhibitory receptors detrimental to the efficacy of

rituximab and trastuzumab (7). Second, a number of studies havedocumented a correlation between the clinical efficacy of Abs inhumans and their allotype of high-affinity (V158) or low-affinity(F158) polymorphic forms of Fc�RIIIa (8–10). Finally, it has beenshown via mutagenesis (11–14) and glycoform engineering (15) thatthe affinity of interaction between Fc and certain Fc�Rs correlateswith cytotoxicity in cell-based assays. Together these data suggestthat an Ab with optimized Fc�R affinity may be more cytotoxicagainst targeted cancer cells in patients (16). For this strategy, thebalance between activating and inhibitory receptors is an importantconsideration, and optimal effector function may result from an Fcwith enhanced affinity for activation receptors and reduced affinityfor the inhibitory receptor Fc�RIIb.

Attempts at improving Ab effector function through mutagenesishave met with incomplete success (14). We used a combination ofcomputational structure-based protein design methods coupledwith high-throughput protein screening to optimize the Fc�Rbinding capacity of Abs. Here we present a series of engineered Fcvariants with improved Fc�R affinity and specificity that provideremarkable enhancements in cytotoxicity.

ResultsDesigned Fc Variants Have Optimized Binding Affinity for Fc�Rs. Acombination of ‘‘directed diversity’’ and ‘‘quality diversity’’ strate-gies were used to computationally optimize the IgG Fc region forFc�R affinity and specificity. Where structural information wasavailable (e.g., the Fc�Fc�RIII complex), we directly optimizedaffinity by designing substitutions that provide more favorableinteractions at the Fc�Fc�R interface. Where structural informa-tion was incomplete or lacking (e.g., the Fc�Fc�RIIb complex),calculations provided a quality set of variants enriched for stabilityand solubility. The advantage of this capability is because of theinnumerable amino acid modifications that are detrimental toproteins (17).

Variants were constructed in the context of the anti-CD52 Abalemtuzumab, expressed and purified, and screened for Fc�Raffinity by using a semiautomated AlphaScreen assay. A number ofengineered Fc variants show significant enhancements in bindingaffinity to human V158 Fc�RIIIa and F158 Fc�RIIIa (Fig. 1A).These variants include the single mutants S239D and I332E and thedouble and triple mutants S239D�I332E and S239D�I332E�A330L[Eu numbering (18)]. The variants show similar levels of enhance-

Conflict of interest statement: G.A.L., W.D., S.K., O.V., J.S.P., L.H., C.C., H.S.C., A.E., S.C.Y.,J.V., D.F.C., R.J.H., and B.I.D. are employees of Xencor. All commercial affiliations, financialinterests, and patent-licensing arrangements that could be considered to pose a financialconflict of interest regarding the submitted article have been disclosed.

This paper was submitted directly (Track II) to the PNAS office.

Freely available online through the PNAS open access option.

Abbreviations: ADCC, Ab-dependent cell-mediated cytotoxicity; ADCP, Ab-dependent cell-mediated phagocytosis; CDC, complement-dependent cytotoxicity; Fc�R, Fc� receptor;LDH, lactate dehydrogenase; NK, natural killer; PBMC, peripheral blood mononuclear cell;SPR, surface plasmon resonance.

*G.A.L., W.D., S.K., and O.V. contributed equally to this work.

†To whom correspondence should be addressed. E-mail: glazar@xencor.com.

© 2006 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0508123103 PNAS � March 14, 2006 � vol. 103 � no. 11 � 4005–4010

APP

LIED

BIO

LOG

ICA

LSC

IEN

CES

ment in the context of the anti-Her2 Ab trastuzumab (Fig. 1B).Similar binding enhancements have been observed in the anti-CD20 Ab rituximab, the anti-EGFR Ab cetuximab, and all otherAbs tested (data not shown). The fits to the binding data providethe inhibitory concentration 50% (IC50) for each Ab, enablingdetermination of the fold-improvements relative to WT (Table 1).Because of the high avidity nature of the assay, the AlphaScreenprovides only relative affinities. True binding constants were ob-tained by using a competition surface plasmon resonance (SPR)experiment (19) in which unbound trastuzumab Ab in an Ab�Fc�Requilibrium was captured to an Fc�RIIIa surface. Initial bindingrates were determined from sensorgram raw data (Fig. 2A), and KDvalues were calculated by plotting the log of receptor concentration

against the initial rate obtained at each concentration (Fig. 2B andTable 1) (20). The WT KD (252 nM) agrees well with published data(208 nM from SPR; 535 nM from calorimetry) (21). KD values ofthe I332E (30 nM) and S239D�I332E (2 nM) variants indicateapproximately one and two logs greater affinity to V158 Fc�RIIIa,respectively.

Binding of the trastuzumab Fc variants to the inhibitory receptorFc�RIIb also was measured by the AlphaScreen (Fig. 1C). Asdiscussed, optimal effector function may result from Fc variants thatprovide greater affinity to activating Fc�Rs relative to the inhibitoryreceptor Fc�RIIb. We refer to this property as a variant’s IIIa:IIbprofile, and define it quantitatively as the fold-Fc�RIIIa affinitydivided by the fold-Fc�RIIb affinity (Table 1). Combination of theA330L mutation with S239D�I332E provides increased Fc�RIIIaaffinity and reduced Fc�RIIb affinity relative to the double variant(Fig. 1 A–C and Table 1), resulting in a significant improvement inIIIa:IIb profile. Enhancements in affinity for the variants also wereobserved for binding to the human activating receptor Fc�RI (datanot shown).

Differences between human and mouse Fc�Rs complicate theuse of mouse cancer models for evaluating the Fc variants in vivo.We investigated the capacity of the double and triple alemtuzumabvariants to enhance affinity to the mouse activating receptorFc�RIII by using the AlphaScreen (Fig. 1D). The data show thatthe S239D�I332E and S239D�I332E�A330L variants also providesignificant improvements in binding to this receptor.

Designed Fc Variants Mediate Enhanced ADCC. Cell-based assays wereperformed to evaluate the capacity of the Fc variants to mediateADCC. Purified human peripheral blood mononuclear cells (PB-MCs) allotyped for the V�F158 Fc�RIIIa polymorphism were usedas effector cells, and lysis was measured by using europium (Eu)-based detection. Trastuzumab Fc variants were tested by using theHer2� breast carcinoma cell line SkBr3 (Fig. 3A). The designed Fcvariants provide substantial ADCC enhancements over WT, and

Fig. 1. Binding of Fc variant Abs to Fc�Rs measured by competitionAlphaScreen. (A) Binding of alemtuzumab Fc variants to human V158 (Left)and F158 (Right) Fc�RIIIa. (B) Binding of trastuzumab Fc variants to humanV158 (Left) and F158 (Right) Fc�RIIIa (n � 2). (C) Binding of trastuzumab Fcvariants to human Fc�RIIb (n � 2). (D) Binding of alemtuzumab variants tomurine Fc�RIII (n � 2). Black asterisk, buffer; gray squares, WT; blackdiamonds, S298A�E333A�K334A; green triangles, S239D; red inverted tri-angles, I332E; blue diamonds, S239D�I332E; and tan circles, S239D�I332E�A330L. The S298A�E333A�K334A variant was generated in a previous study(14) and is used here as comparison.

Table 1. Fc�R affinity enhancements of Fc variants

Variant

Alem AS [LOG(IC50) (M)] fold Tras AS [LOG(IC50) (M)] fold Tras SPR [KD] fold

V158 IIIa F158 IIIa V158 IIIa F158 IIIa IIb IIIa:IIb* V158 IIIa

WT [�7.60 � 0.02] 1 [�6.90 � 0.06] 1 [�6.42 � 0.06] 1 [�6.61 � 0.05] 1 [�7.23 � 0.07] 1 1 [252 � 89 nM] 1S298A�E333A�K334A† [�8.71 � 0.13] 13 [�8.01 � 0.10] 13S239D [�8.72 � 0.12] 13 [�7.72 � 0.06] 7 [�7.65 � 0.06] 17 [�7.55 � 0.06] 9 [�8.06 � 0.07] 7 2I332E [�8.61 � 0.08] 10 [�7.89 � 0.09] 10 [�7.22 � 0.05] 6 [�7.23 � 0.07] 4 [�8.00 � 0.06] 6 1 [30 � 7 nM] 8S239D�I332E [�9.44 � 0.08] 70 [�8.70 � 0.10] 63 [�8.83 � 0.05] 254 [�8.10 � 0.06] 31 [�9.07 � 0.05] 69 4 [2 � 2 nM] 126S239D�I332E�A330L [�9.66 � 0.07] 115 [�9.12 � 0.05] 169 [�8.99 � 0.05] 370 [�8.38 � 0.08] 58 [�8.84 � 0.07] 41 9

Alemtuzumab (Alem) and trastuzumab (Tras) AlphaScreen (AS) or SPR data provide LOG(IC50) or KD [bracketed] values followed by folds relative to WT. Fold �IC50variant�IC50WT.*IIIa:IIb � fold V158 Fc�RIIIa�fold Fc�RIIb for trastuzumab.†Generated in a previous study (14) and used here for comparison.

Fig. 2. Affinity of Fc variant trastuzumab Abs for V158 Fc�RIIIa measured bycompetition SPR. (A) Sensorgrams showing binding to an Fc�RIIIa surface byunbound S239D�I332E Ab in an Ab�Fc�RIIIa equilibrium at increasing receptorconcentrations. (B) Plot of normalized initial binding rate vs. log of receptorconcentration. Derived KD values are presented in Table 1. Line colors are asfollows: gray, WT; red, I332E; and blue, S239D�I332E.

4006 � www.pnas.org�cgi�doi�10.1073�pnas.0508123103 Lazar et al.

the relative ADCC enhancements are proportional to theirFc�RIIIa affinities. Between two and three logs improvement inpotency are observed for all three allelic forms, reflected in shiftsin EC50 (effective concentration 50%) toward lower concentra-tions. Additionally, shifts are observed toward higher maximallevels of ADCC at saturating concentrations, reflecting improve-ments in the relative efficacy of the variants. Substantial enhance-ments also are observed for the Fc variants in the context ofalemtuzumab (Fig. 3B) and rituximab (Fig. 3C), using release oflactate dehydrogenase (LDH) as the detection method. The vari-ants provide comparable enhancements in all other Abs tested(data not shown), again consistent with the context-independenceof the improvements.

Designed Fc Variants Mediate Enhanced ADCC Across a Range ofAntigen Expression Levels. A critical parameter governing the clin-ical efficacy of anticancer Abs is the expression level of targetantigen. Indeed, clinical trials of trastuzumab have been limited topatients showing a moderate (2�) to strong (3�) level of Her2overexpression by immunohistochemistry (22), reflecting �500,000and 2,300,000 receptors per cell respectively (23). To explore thecytotoxic capacity of our Fc variants at different antigen expressionlevels, ADCC was measured for WT and variant trastuzumab Absagainst four different cell lines expressing amplified to low levels ofHer2 (Fig. 4A). The S239D�I332E and S239D�I332E�A330L vari-ants provide substantial ADCC enhancements over WT trastu-zumab across a broad range of antigen expression level (Fig. 4B).In addition, at barely observable antigen expression and WT ADCClevels (the MCF7 cell line), ADCC using the variant Abs isimproved above the detectable threshold.

Enhanced Effector Function Is Mediated by Multiple Effector Cells. Toexplore the specific cell types involved in target cell lysis, the rolesof two PBMC components, natural killer (NK) cells and macro-phages, were investigated. NK cells express only the activatingreceptors Fc�RIIIa and in some cases Fc�RIIc (24). Substantialcytotoxicity enhancements are observed by using NK cells aseffector cells, including enhancements in both EC50 and in maximallysis (Fig. 5A). Phagocytes such as macrophages, neutrophils, anddendritic cells express both activating and inhibitory Fc�Rs, andFc�R-mediated phagocytosis of target cells may result in acutecytotoxicity and promotion of adaptive immunity (25). Fluores-cence imaging of differentially labeled macrophages and Her2�

target cells after coculture in the presence of S239D�I332E tras-tuzumab results in visible engulfment (Fig. 5B). Quantitative mea-surement of phagocytosis using flow cytometry indicates that theS239D�I332E�A330L variant provides a subtle yet significant en-hancement in ADCP relative to WT trastuzumab at higher con-centrations (Fig. 5C). A similar experiment in the context ofrituximab shows ADCP enhancement for the S239D�I332E andS239D�I332E�A330L variants against CD20� cells (Fig. 5D). Thereason for the reduced ADCP of the triple variant at higherconcentrations is unknown.

Designed Fc Variants Show Differential Capacity to Mediate Comple-ment-Dependent Cytotoxicity (CDC). CDC is another effector func-tion by which some Abs may destroy tumor cells. Interaction of Abwith complement is mediated by the protein C1q, the binding sitefor which is separate from but overlapping with the Fc�R site (26,27). Alamar Blue release was used to monitor lysis of Fc variant andWT rituximab-opsonized CD20� target cells by human serumcomplement. Whereas S239D�I332E rituximab elicits CDC com-parable with WT, the addition of A330L ablates CDC (Fig. 6). Thisresult is not surprising given the proximity of A330 to the C1qbinding site (26, 27). The set of S239D�I332E and S239D�I332E�A330L variants thus provide the option for enhancing ADCC incases where CDC is desired or undesired. Notably, other substitu-tions at position 330 provide similar enhancements in Fc�RIIIaaffinity and IIIa:IIb profile yet do not affect CDC (data not shown).

Designed Fc Variants Mediate Enhanced B Cell Depletion in Macaques.Peripheral B cell depletion by rituximab in cynomolgus monkeyshas been reported as a suitable measure of anti-CD20 cytotoxicity(28). The advantage of this system is that monkey Fc�Rs, in contrast

Fig. 3. Cell-based ADCC assays of Fc variant Abs. (A) Eu-based detectionassays of trastuzumab Abs against SkBr3 breast carcinoma target cells in thepresence of human PBMCs allotyped for V�V (Top), V�F (Middle), or F�F(Bottom) 158 Fc�RIIIa. (B) LDH-based detection assay of alemtuzumab Absagainst DoHH-2 lymphoma target cells in the presence of human PBMCsallotyped for F�F158 Fc�RIIIa. (C) LDH-based detection assay of rituximab Absagainst WIL2-S lymphoma target cells in the presence of human PBMCsallotyped for F�F158 Fc�RIIIa. n � 2 for all assays. Gray squares, WT; blackdiamonds, S298A�E333A�K334A (14); green triangles, S239D; red invertedtriangles, I332E; blue diamonds, S239D�I332E; and tan circles, S239D�I332E�A330L.

Fig. 4. Cell-based ADCC assay of trastuzumab Fc variants against cell linesexpressing varying levels of Her2 receptor. (A) Western blot showing expres-sion levels of Her2 for cell lines SkBr3 (�106 copies per cell), SkOV3 (�105 copiesper cell), OVCAR3 (�104 copies per cell), and MCF7 (�102 to 103 copies per cell).Specified cancer cells were washed in PBS and resuspended at 1 � 105 cells perml in lysis buffer. Equivalent amounts of each lysate were loaded per well ofa SDS�PAGE gel, followed by Western analysis using 2 �g�ml commercialtrastuzumab and 1 �g�ml horseradish peroxidase-conjugated secondary Ab.(B) ADCC of trastuzumab Fc variants against the Her2� cell lines in thepresence of human PBMCs (F�F158 Fc�RIIIa). Ab concentration was 1 ng�ml,and lysis was measured by using Eu-based detection. Data were normalized tothe minimum and maximum fluorescence signal provided PBMCs alone andTriton X-100, respectively (n � 3). Bar colors are as follows: gray, WT trastu-zumab; blue, S239D�I332E; and tan, S239D�I332E�A330L.

Lazar et al. PNAS � March 14, 2006 � vol. 103 � no. 11 � 4007

APP

LIED

BIO

LOG

ICA

LSC

IEN

CES

to those in mice, are highly homologous to human receptors. Fourvariant and two WT doses were evaluated to approximate the doserequired to deplete 50% of circulating B cells. An enhanced levelof B cell depletion is observed for the S239D�I332E variant relativeto WT as measured by the population of CD20� (Fig. 7A) andCD40� (Fig. 7B) cells. Both variant and WT show a characteristicrebound in B cells (28) followed by further reduction and gradualrecovery, with the greatest level of depletion occurring at day 5. Bcell level was not yet fully recovered at 28 days but returned topredose levels by day 84 (data not shown). Interpolation of the day5 data at the approximate dose required for 50% B cell depletionsuggests a dose of nearly 10 �g�kg per day for WT, in goodagreement with historical data (28). For the S239D�I332E variant,

a dose of 0.2 �g�kg per day is sufficient for 50% depletion (Fig. 7C),an apparent �50-fold increase in potency. Concerns about thepotential for Ab�Fc�RIIIa interactions to promote apoptosis ofactivated NK cells (29) also led us to investigate the effect of thevariant rituximab on NK cell levels. A dose-dependent decrease inNK cells is observed in all groups as measured by the population ofCD3��CD16� (Fig. 7D) cells, correlated with the degree of B celldepletion effected. No difference is observed in the relative reduc-tion of NK cells compared with B cells between WT and variantanti-CD20 Abs. NK cell populations recovered to predose rangewithin 2 weeks of the initial dose. Identical NK cell results wereobtained monitoring CD3��CD8� cells (data not shown). Nosignificant changes were observed in monocytes, T helper lympho-cytes, T cytotoxic�suppressor lymphocytes, or total T lymphocytesas measured by the populations of CD3��CD14�, CD3��CD4�,CD3��CD8�, and CD3� cells, respectively (data not shown).

DiscussionWe have capitalized on recent advances in protein engineering togenerate a series of Fc variants with optimized Fc�R affinity,enhanced effector function in cell-based assays, and increasedefficacy in a preclinical animal system. Our method relies heavily onstate-of-the-art computer algorithms to search sequence-structurespace, semiautomated protein expression and purification, and ahigh-throughput primary screen that is extremely reliable at iden-tifying variants with desirable properties.

There were several engineering challenges in this study, includingthe essentially identical binding sites on Fc for the activating andinhibitory receptors, the proximal but overlapping putative bindingsite for C1q, and the fact that homodimeric Fc binds asymmetricallyto monomeric Fc�R. A consequence of this latter issue is that adesigned substitution is effectively two mutations, one on each sideof the interface yet in distinct structural environments. Thus,substitutions that result in overall enhanced receptor affinity arethose in which the sum of interactions at both interfaces is ener-getically beneficial. Detailed structural analysis of the mutations isprovided in Supporting Text and Fig. 8, which are published assupporting information on the PNAS web site.

Fig. 5. Role of NK cells and macrophages in mediating enhanced effectorfunction. (A) Cell-based ADCC assay of variant trastuzumab Abs against SkBr3breast carcinoma target cells in the presence of human F�F158 Fc�RIIIa NKcells. Lysis was measured by using LDH-based detection. Gray squares, WTtrastuzumab; green triangles, S239D; red inverted triangles, I332E; blue dia-monds, S239D�I332E; and tan circles, S239D�I332E�A330L. (B) Dual fluores-cence merge image of PKH67-labeled SkBr3 cells (green) and RPE-anti-CD11�anti-CD14-labeled macrophages (red) after a 24-h coculture (3:1macrophage:SkBr3 ratio) in the presence of 100 ng�ml S239D�I332E. Phago-cytosis of green target cells within red-labeled macrophages is detected asyellow in the merge image. (C) ADCP enhancement of Fc variant trastuzumabAb against SkBr3 target cells, measured using flow cytometry. (D) ADCPenhancement of Fc variant rituximab Abs against WIL2-S target cells. % ADCPrepresents the number of colabeled cells (macrophage plus target) over thetotal number of target cells in the population (phagocytosed plus nonphago-cytosed) after 10,000 counts. Bar colors are as follows: black, buffer; gray, WTtrastuzumab; blue, S239D�I332E; and tan, S239D�I332E�A330L. (n � 2 for allassays.)

Fig. 6. Cell-based CDC assay of rituximab Fc variants. Lysis of WIL2-S lym-phoma target cells in the presence of human complement was measured byusing Alamar Blue release (n � 2). Gray squares, WT rituximab; black dia-monds, S239D�I332E; and black circles, S239D�I332E�A330L.

Fig. 7. B cell depletion in macaques. (A) Percent CD20� B cells remainingduring treatment with WT and S239D�I332E rituximab Abs. (B) Percent CD40�

B cells remaining during treatment. (C) Dose-response of CD20� B cell levels totreatment with S239D�I332E rituximab, acquired at day 5. (D) Percent CD3��CD16� NK cells remaining during treatment. Data reported are group aver-ages of three monkeys per treatment group (n � 3). Symbols and experimen-tally determined doses are as follows: filled gray squares, WT rituximab (2�g�kg), open gray squares, WT rituximab (34 �g�kg); and black diamonds,S239D�I332E (2 �g�kg).

4008 � www.pnas.org�cgi�doi�10.1073�pnas.0508123103 Lazar et al.

The variants with the greatest enhancements in Fc�RIIIa affinityalso significantly increase binding to Fc�RIIb. The addition ofA330L to S239D�I332E provides a moderate but significant im-provement in IIIa:IIb profile. The simultaneous Fc�RIIIa affinityimprovement of the triple variant over the double and its subtle butquestionable ADCC enhancement make it difficult to draw con-clusions about the sufficiency of the improved specificity andrelated impact on in vitro effector function. The relevance ofreceptor selectivity is speculative, based primarily on improvedantitumor efficacy in Fc�RII-deficient mice (7). Theoretically theoptimal variant with respect to human receptors is selective forFc�RIIa and Fc�RIIc over Fc�RIIb.

Affinity was improved for both the V158 and F158 forms ofFc�RIIIa, and ADCC enhancements were observed using PBMCsfrom donors homozygous for both allelic forms of the receptor. Theclinical relevance of the V�F158 polymorphism is well supported (8,9), and given the predominance of F158 in the population (�20%V�V, 40% V�F, and 40% F�F), affinity for this low-affinity�low-responder receptor is an important clinical parameter. Notably,affinities of the best variants for F158 Fc�RIIIa are significantlybetter than that of WT for the V158 isoform, inferred from theAlphaScreen data. This result suggests that the variants may enablethe clinical efficacy of Abs for the less-responsive patient populationto achieve that currently possible for high responders (8–10).Together the results indicate that the Fc variants will be broadlyapplicable to the entire patient population and that clinical im-provement will potentially be greatest for the less-responsive pa-tients who need it most.

Results from the cell-based assays show that both NK cells andmacrophages are active PBMC components in the enhanced ef-fector function of the Fc variants. Although not well characterized,the relative importance of different cell types in Ab therapy is likelycancer-dependent, due to a minimum to differences in tumorlocation�accessibility and the particular immune state of a givenpatient. It follows that with regard to effector function it may besensible to separate cancers into hematologic and solid tumors. Thepresence of NK cells in peripheral blood and their capacity forFc�RIIIa-mediated target cell lysis are logical bases of support forthe importance of NK cells in rituximab-mediated lymphomacytotoxicity. The role of other effector cells is supported by theenhanced rituximab protection in Fc�RII�/� mice (7), in agree-ment with the Fc�R-dependent role of macrophages observed inthe ADCP assay of the present study. The contribution of ADCPto Ab therapy may be twofold. Engulfment can result in immediatedestruction of target cells, akin to ADCC. Additionally, Fc�R-mediated phagocytosis and endocytosis are mechanisms of antigenuptake, potentially leading to antigen presentation and adaptiveimmunity (25). Fc�R- and Ab-mediated dendritic cell maturation,antigen presentation, and antitumor T cell immunity have beendemonstrated in mice (30–32) and by human cells in vitro (33–35).Nonetheless, the role of adaptive immunity in mAb therapy remainsunclear. In addition to their potential for improving clinical out-come, our Fc variants will be a unique set of reagents with whichto investigate the relevance of different receptors, cell types,effector functions, and immune responses to Ab mechanism ofaction.

Our B cell depletion experiments deliberately focused on themacaque system to maintain a high degree of homology withhuman immune biology. The S239D�I332E variant clearly showsincreased potency relative to WT rituximab, consistent with itsenhanced receptor affinity and ADCC in vitro, and with theobservation that B cell depletion by rituximab in vivo is dominatedby Fc�R-mediated mechanisms (36, 37). A number of factors mayaffect in vivo performance, including the high concentration ofnonspecific IgG in serum (37, 38). The macaque experiment wasundertaken because, short of a clinical trial, it is the best predictorof clinical effect. Accordingly, the capacity of the engineered Fc

region to substantially enhance efficacy in the current model issignificant motivation for its use in clinical trials.

Together the improvements in effector function, the enhance-ment for the more common yet less responsive F158 Fc�RIIIaallele, and the greater killing capacity at lower antigen expressionlevels have promising implications for mAb therapy. Most evidentis the possibility of expanding the population of patients responsiveto treatment. An equally profound implication is the potentialimpact on the clinical utility of targets. The ability to target a giventumor antigen depends on an array of parameters including ex-pression level, expression profile, structural and spatial organiza-tion in the membrane, requirement for growth or metastasis, andcapacity to signal. These latter two factors have thus far been themost indicative of clinical response; all currently approved antican-cer Abs perturb growth or signaling in some manner, either byblocking activation or accelerating turnover of a needed growthfactor receptor or by eliciting apoptotic signaling events. Yet someantigens that show promising differential expression, for examplemucins and adhesion proteins, may serve merely as ‘‘tumor han-dles’’ for the immune system. Overexpression of such proteins ontumors is perhaps related more to their role in metastasis orimmune evasion rather than conferral of some proliferative advan-tage. Despite the number of approved Abs with the capacity toinhibit growth or signaling, it may not be a requisite for success.Rather, inability to directly affect growth may mean that an Abmust rely more heavily on mechanisms of action that involveengagement of the immune system, namely Fc�R- and comple-ment-mediated cytotoxicity. The improvements observed for the Fcvariants described here are a significant step toward enabling thetargeting of a broader and more diverse set of tumor antigens.

Materials and MethodsProtein Design. Design calculations were carried out by usingProtein Design Automation technology (39) and Sequence Predic-tion Algorithm technology (40). Detailed description is provided inSupporting Text.

Construction, Expression, and Purification of Ab Variants and Fc�Rs.Variant Abs were constructed by using quick-change mutagenesis,expressed in 293T cells, and purified by using protein A chroma-tography. Fc�Rs were constructed as C-terminal �6xHis-GSTfusions, expressed in 293T (human Fc�Rs) or NIH 3T3 (mouseFc�RIII) cells, and purified by using nickel affinity chromatogra-phy. Detailed description is provided in Supporting Text.

Binding Assays. AlphaScreen assays used untagged Ab to competethe interaction between biotinylated IgG bound to streptavidindonor beads and Fc�R-His-GST bound to anti-GST acceptorbeads. Competition SPR (19) experiments measured capture offree Ab from a preequilibrated Ab�receptor analyte mixture toV158 Fc�RIIIa-His-GST bound to an immobilized anti-GST sur-face. Equilibrium dissociation constants (KD values) were calcu-lated by using the proportionality of initial binding rate on free Abconcentration in the Ab�receptor equilibrium (20). Detailed de-scription of AlphaScreen and SPR assays is provided in SupportingText.

Cell-Based Assays. ADCC was measured by using either theDELFIA EuTDA-based cytotoxicity assay (PerkinElmer) or theLDH Cytotoxicity Detection Kit (Roche Diagnostics). HumanPBMCs were purified from leukopacks by using a Ficoll gradientand allotyped for V�F158 Fc�RIIIa by using PCR (41). NK cellswere isolated from human PBMCs by using negative selection andmagnetic beads (Miltenyi Biotec, Auburn, CA). Target cell lineswere obtained from American Type Culture Collection. For Eu-based detection, target cells were first loaded with BATDA[Bis(acetoxymethyl)-2,2�:6�,2�-terpyridine-6,6�-dicarboxylate] at1 � 106 cells per ml and washed 4�. For both Eu- and LDH-based

Lazar et al. PNAS � March 14, 2006 � vol. 103 � no. 11 � 4009

APP

LIED

BIO

LOG

ICA

LSC

IEN

CES

detection, target cells were seeded into 96-well plates at 10,000 cellsper well, and opsonized by using Fc variant or WT Abs at theindicated final concentration. Triton X-100 and PBMCs alone wererun as controls. Effector cells were added at 25:1 PBMCs:targetcells or 4:1 NK cells:target cells, and the plate was incubated at 37°Cfor 4 h. Cells were incubated with either Eu3� solution or LDHreaction mixture, and fluorescence was measured by using a FusionAlpha-FP (PerkinElmer). Data were normalized to maximal (Tri-ton) and minimal (PBMCs alone) lysis and fit to a sigmoidaldose-response model.

For phagocytosis experiments, monocytes were isolated fromhuman V�F158 Fc�RIIIa PBMCs by using a Percoll gradient anddifferentiated into macrophages by culture with 0.1 ng�ml granu-locyte�macrophage colony-stimulating factor for 1 week. For im-aging, SkBr3 target cells were labeled with PKH67 (Sigma) andcocultured for 24 h with macrophages at a 3:1 effector:target cellratio in the presence of 100 ng�ml S239D�I332E trastuzumab. Cellsthen were treated with secondary Abs anti-CD11-RPE and anti-CD14-RPE (DAKO) for 15 min before live cell imaging using aNikon Eclipse TS100 fluorescence microscope. For quantitativeADCP, target cells (SkBr3 for trastuzumab and WIL2-S for ritux-imab) were labeled with PKH67, seeded in a 96-well plate at 20,000cells per well, and treated with WT or variant Ab at the designatedfinal concentrations. Macrophages were labeled with PKH26(Sigma) and added to the opsonized labeled target cells at 20,000cells per well, and the cells were cocultured for 18 h. Fluorescencewas measured by using dual-label flow cytometry at the City ofHope Flow Cytometry Unit (Duarte, CA).

For CDC assays, target WIL2-S lymphoma cells were washed 3�in 10% FBS medium by centrifugation and resuspension andseeded at 50,000 cells per well. WT or variant rituximab Ab wasadded at the indicated final concentrations. Human serum com-plement (Quidel, San Diego) was diluted 50% with medium andadded to Ab-opsonized target cells. Final complement concentra-

tion was one-sixth original stock. Plates were incubated for 2 h at37°C, Alamar Blue was added, cells were cultured for 2 days, andfluorescence was measured. Data were normalized to the maxi-mum and minimum signal and fit to a sigmoidal dose-responsecurve.

In Vivo B Cell Depletion. Monkey studies were conducted at CharlesRiver Laboratories, Sierra Biomedical Division. Cynomolgus mon-keys (Macaca fascicularis) were injected i.v. once daily for 4consecutive days with WT or S239D�I332E rituximab Ab. Theexperiment comprised six treatment groups of �0.2, 2, 7, or 34�g�kg (S239D�I332E) or �2 or 34 �g�kg (WT control), with threemonkeys per treatment group. Blood samples were acquired on twoseparate days before dosing (baseline) and at days 1, 2, 5, 15, and28 after initiation of dosing. For each sample, cell populations werequantified with flow cytometry by using specific Abs against thefollowing marker antigens: CD2��CD20� (all lymphocytes, samplepurity�total B cells), CD20� and CD40� (B lymphocytes), CD3� (Tlymphocytes), CD3��CD4� (T helper lymphocytes), CD3��CD8�

(T cytotoxic�suppressor lymphocytes), CD3��CD16� and CD3��CD8� (NK cells), and CD3��CD14� (monocytes). Absolute num-bers of each cell type were determined by multiplying the propor-tion of cells expressing the indicated markers by the absolutelymphocyte count and�or absolute monocyte count (determined bystandard hematological analysis). Percent B cell depletion wascalculated by comparing B cell counts on the given day with theaverage of the two baseline measures for each animal.

We thank John Desjarlais, Steve Doberstein, and Art Chirino forintellectual contributions; Meridith Alden for assistance with DNAsequencing; and Marie Ary for help with the manuscript. We thankLucy Brown (City of Hope Flow Cytometry Unit) for her assistance andPatrick Lappin (Charles River Laboratories) for guidance with themacaque study.

1. Weiner, L. M. & Carter, P. (2005) Nat. Biotechnol. 23, 556–557.2. Cohen-Solal, J. F., Cassard, L., Fridman, W. H. & Sautes-Fridman, C. (2004)

Immunol. Lett. 92, 199–205.3. Jefferis, R. & Lund, J. (2002) Immunol. Lett. 82, 57–65.4. Raghavan, M. & Bjorkman, P. J. (1996) Annu. Rev. Cell Dev. Biol. 12, 181–220.5. Sondermann, P., Kaiser, J. & Jacob, U. (2001) J. Mol. Biol. 309, 737–749.6. Radaev, S. & Sun, P. (2002) Mol. Immunol. 38, 1073–1083.7. Clynes, R. A., Towers, T. L., Presta, L. G. & Ravetch, J. V. (2000) Nat. Med.

6, 443–446.8. Cartron, G., Dacheux, L., Salles, G., Solal-Celigny, P., Bardos, P., Colombat,

P. & Watier, H. (2002) Blood 99, 754–758.9. Weng, W. K. & Levy, R. (2003) J. Clin. Oncol. 21, 3940–3947.

10. Dall’Ozzo, S., Tartas, S., Paintaud, G., Cartron, G., Colombat, P., Bardos, P.,Watier, H. & Thibault, G. (2004) Cancer Res. 64, 4664–4669.

11. Duncan, A. R., Woof, J. M., Partridge, L. J., Burton, D. R. & Winter, G. (1988)Nature 332, 563–564.

12. Sarmay, G., Lund, J., Rozsnyay, Z., Gergely, J. & Jefferis, R. (1992) Mol.Immunol. 29, 633–639.

13. Redpath, S., Michaelsen, T. E., Sandlie, I. & Clark, M. R. (1998) Hum.Immunol. 59, 720–727.

14. Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G., Rae, J., Briggs, J., Xie,D., Lai, J., Stadlen, A., Li, B., et al. (2001) J. Biol. Chem. 276, 6591–6604.

15. Shields, R. L., Lai, J., Keck, R., O’Connell, L. Y., Hong, K., Meng, Y. G.,Weikert, S. H. & Presta, L. G. (2002) J. Biol. Chem. 277, 26733–26740.

16. Cartron, G., Watier, H., Golay, J. & Solal-Celigny, P. (2004) Blood 104, 2635–2642.17. Marshall, S. A., Lazar, G. A., Chirino, A. J. & Desjarlais, J. R. (2003) Drug

Discov. Today 8, 212–221.18. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. & Foeller, C. (1991)

Sequences of Proteins of Immunological Interest (U.S. Dept. of Health and Hum.Serv., Bethesda).

19. Nieba, L., Krebber, A. & Pluckthun, A. (1996) Anal. Biochem. 234, 155–165.20. Edwards, P. R. & Leatherbarrow, R. J. (1997) Anal. Biochem. 246, 1–6.21. Okazaki, A., Shoji-Hosaka, E., Nakamura, K., Wakitani, M., Uchida, K.,

Kakita, S., Tsumoto, K., Kumagai, I. & Shitara, K. (2004) J. Mol. Biol. 336,1239–1249.

22. Vogel, C. L., Cobleigh, M. A., Tripathy, D., Gutheil, J. C., Harris, L. N.,Fehrenbacher, L., Slamon, D. J., Murphy, M., Novotny, W. F., Burchmore, M.,et al. (2002) J. Clin. Oncol. 20, 719–726.

23. Ross, J. S., Fletcher, J. A., Linette, G. P., Stec, J., Clark, E., Ayers, M.,Symmans, W. F., Pusztai, L. & Bloom, K. J. (2003) Oncologist 8, 307–325.

24. Ernst, L. K., Metes, D., Herberman, R. B. & Morel, P. A. (2002) J. Mol. Med.80, 248–257.

25. Amigorena, S. & Bonnerot, C. (1999) Semin. Immunol. 11, 385–390.26. Thommesen, J. E., Michaelsen, T. E., Loset, G. A., Sandlie, I. & Brekke, O. H.

(2000) Mol. Immunol. 37, 995–1004.27. Idusogie, E. E., Presta, L. G., Gazzano-Santoro, H., Totpal, K., Wong, P. Y.,

Ultsch, M., Meng, Y. G. & Mulkerrin, M. G. (2000) J. Immunol. 164,4178–4184.

28. Reff, M. E., Carner, K., Chambers, K. S., Chinn, P. C., Leonard, J. E., Raab,R., Newman, R. A., Hanna, N. & Anderson, D. R. (1994) Blood 83, 435–445.

29. Warren, H. S. & Kinnear, B. F. (1999) J. Immunol. 162, 735–742.30. Regnault, A., Lankar, D., Lacabanne, V., Rodriguez, A., Thery, C., Rescigno,

M., Saito, T., Verbeek, S., Bonnerot, C., Ricciardi-Castagnoli, P. & Amigorena,S. (1999) J. Exp. Med. 189, 371–380.

31. Rafiq, K., Bergtold, A. & Clynes, R. (2002) J. Clin. Invest. 110, 71–79.32. Kalergis, A. M. & Ravetch, J. V. (2002) J. Exp. Med. 195, 1653–1659.33. Dhodapkar, K. M., Krasovsky, J., Williamson, B. & Dhodapkar, M. V. (2002)

J. Exp. Med. 195, 125–133.34. Dhodapkar, M. V., Krasovsky, J. & Olson, K. (2002) Proc. Natl. Acad. Sci. USA

99, 13009–13013.35. Groh, V., Li, Y. Q., Cioca, D., Hunder, N. N., Wang, W., Riddell, S. R., Yee,

C. & Spies, T. (2005) Proc. Natl. Acad. Sci. USA 102, 6461–6466.36. Uchida, J., Hamaguchi, Y., Oliver, J. A., Ravetch, J. V., Poe, J. C., Haas, K. M.

& Tedder, T. F. (2004) J. Exp. Med. 199, 1659–1669.37. Vugmeyster, Y. & Howell, K. (2004) Int. Immunopharmacol. 4, 1117–

1124.38. Preithner, S., Elm, S., Lippold, S., Locher, M., Wolf, A., Silva, A. J., Baeuerle,

P. A. & Prang, N. S. (2006) Mol. Immunol. 43, 1183–1193.39. Dahiyat, B. I. & Mayo, S. L. (1996) Protein Sci. 5, 895–903.40. Raha, K., Wollacott, A. M., Italia, M. J. & Desjarlais, J. R. (2000) Protein Sci.

9, 1106–1119.41. Leppers-van de Straat, F. G., van der Pol, W. L., Jansen, M. D., Sugita, N.,

Yoshie, H., Kobayashi, T. & van de Winkel, J. G. (2000) J. Immunol. Methods242, 127–132.

4010 � www.pnas.org�cgi�doi�10.1073�pnas.0508123103 Lazar et al.