Monoclonal Antibody to Human CD4 Effector Functions of a Modified

Home , Clenoliximab, Fc receptor, Keliximab

Elimination of Fc Receptor-Dependent Effector Functions of a Modified IgG4 Monoclonal Antibody to Human CD4

This information is current as Manjula P. Reddy, Cheryl Ann S. Kinney, Margery A. of September 28, 2021. Chaikin, Angela Payne, Jacqueline Fishman-Lobell, Ping Tsui, Paul R. Dal Monte, Michael L. Doyle, Michael R. Brigham-Burke, Darrell Anderson, Mitchell Reff, Roland Newman, Nabil Hanna, Raymond W. Sweet and Alemseged Truneh Downloaded from J Immunol 2000; 164:1925-1933; ; doi: 10.4049/jimmunol.164.4.1925 http://www.jimmunol.org/content/164/4/1925 http://www.jimmunol.org/ References This article cites 38 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/164/4/1925.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Elimination of Fc Receptor-Dependent Effector Functions of a Modiﬁed IgG4 Monoclonal Antibody to Human CD4

Manjula P. Reddy,* Cheryl Ann S. Kinney,* Margery A. Chaikin,1* Angela Payne,† Jacqueline Fishman-Lobell,‡ Ping Tsui,* Paul R. Dal Monte,† Michael L. Doyle,§ Michael R. Brigham-Burke,§ Darrell Anderson,¶ Mitchell Reff,¶ Roland Newman,¶ Nabil Hanna,¶ Raymond W. Sweet,* and Alemseged Truneh2*

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque

mAb to human constant regions. The heavy chain constant region is a modiﬁed IgG4 containing two single residue substitutions Downloaded from designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells,

except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory http://www.jimmunol.org/ attributes of keliximab without the liability of strong Fc␥ receptor binding. In initial clinical trials, these properties have translated .to a reduced incidence of CD4؉ T cell depletion. The Journal of Immunology, 2000, 164: 1925–1933

D4 is a cell surface glycoprotein expressed on T lineage ise for the treatment of RA and other autoimmune disorders (6–9). cells, including the majority of thymocytes, a subset of However, the use of murine CD4 mAbs in immunocompetent C peripheral T cells, and also at low level on monocytes. hosts is limited by the human anti-mouse Ab response that clears On mature T cells, CD4 serves a corecognition function through the infused mAb and may lead to anaphylactic responses (7). Sev- interaction with MHC class II molecules expressed on APC. CD4ϩ eral strategies have emerged to minimize the immunogenicity of

T cells constitute primarily the helper subset which regulate T and mAbs. One approach is primitization in which V regions from Abs by guest on September 28, 2021 B cell functions during T-dependent responses to viral, bacterial, generated in macaques are fused to human constant domains (10). fungal, or parasitic infections (1, 2). A second issue for CD4 mAbs is Ab-mediated depletion of CD4 In the pathogenesis of autoimmune diseases, such as rheumatoid cells, particularly for chronic therapy, where prolonged depression arthritis (RA),3 CD4ϩ T cells foster and aggravate inflammatory of CD4 cells may compromise the immune status of patients. For conditions resulting in tissue destruction (3). One strategy to in- example, severe and prolonged depletion of CD4 cells was ob- tervene in these diseases is to short-circuit the inflammatory con- served with the murine-human chimeric mAb (cM-T412) even dition by denying T cells the capacity to be continuously activated with a single dose (11). Several studies in animal models indicate and thereby returning the immune system to a state of self-toler- that CD4 cell depletion is not required, or even preferred, for im- ance (4). This approach requires specific targets for intervention munosuppression by CD4 mAbs. Abs devoid of cell-depleting po- Ј with a high degree of efficacy and low toxicity or side effects (5). tential, including F(ab )2 fragments block immune responses to One such treatment strategy is the use of CD4 mAbs for the se- coadministered Ags and prevent the development of spontaneous ϩ lective inactivation or removal of CD4 T cells. Results from ini- autoimmune conditions in genetically susceptible mice (12–14). tial clinical trials with murine mAbs to human CD4 showed prom- Moreover, treatment of these mice did not impair long-term immunological memory because response to recall Ags was unaffected. Departments of *Immunology, †Drug Metabolism and Pharmacokinetics, ‡Oncology, Keliximab (CE9.1 or SB-210396) is a primatized CD4 mAb and §Structural Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA with human ␭ and ␥1 constant regions (15). This Ab binds with 19406; and ¶IDEC Pharmaceuticals Corporation, San Diego, CA 92121 high affinity to human and chimpanzee CD4, induces CD4 receptor Received for publication June 21, 1999. Accepted for publication December 3, 1999. down modulation, and is a potent inhibitor of in vitro T cell re- The costs of publication of this article were defrayed in part by the payment of page sponses (15). As predicted for an IgG1 isotype Ab, keliximab charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. showed efficient binding to human IgG Fc receptors. Unexpect- 1 Current address: 3-Dimensional Pharmaceuticals, 665 Stockton Drive, Suite 104, edly, this Ab did not bind to complement component C1q or me- Exton, PA 19341. diate complement-dependent cytotoxicity, and in vivo in chimpan- 2 Address correspondence and reprint requests to Dr. Alemseged Truneh, Mail Code zees showed only modest depletion of CD4 cells even upon repeat UW2101, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box administration. Because of these unusual but desirable properties, 1539, King of Prussia, PA, 19406. E-mail address: [email protected] keliximab entered human clinical trials for the treatment of RA and 3 Abbreviations used in this paper: RA, rheumatoid arthritis; sCD4, soluble CD4 Ag; RF, rheumatoid factor; CDC, complement dependent cytotoxicity; ADCC, Ab-de- asthma. Nonetheless, treatment with keliximab lead to a variable pendent cell-mediated cytotoxicity; MFI, mean fluorescent intensity. extent and duration of CD4 cell depletion (16).

FIGURE 1. Schematic representation of keliximab and clenoliximab. Both are Cynomolgus macaque/human chimeric mAbs, containing identical heavy and light chain variable regions. They also contain the same light chain constant domains, differing only in the heavy chain constant region. Keliximab is of IgG1 isotype and clenoliximab is of modified IgG4 isotype with two amino acid substitutions as shown above. Residues 227–230 represent 239–242 and residues 235–240 represent 247–252 in the Kabat numbering system (17). Downloaded from During the progression of keliximab, we developed clenolix- from Anex1 into the expression vector NEOSPLA 4 and produced in DG44 imab, a modified IgG4 derivative of keliximab designed to mini- CHO cells and purified essentially as described for keliximab (15). ␥ ϩ mize cell depletion. The 4 constant region contains two amino Binding to CD4 T cells ␥ acid substitutions relative to native 4. Substitution of glutamic ϩ acid for leucine at residue 235 (EU numbering, Ref. 17) in the CH2 A subclone of the CD4 thymocyte cell line SupT1 (SupT1-18; enriched ␥ from SupT1 by FACS for high CD4 expression) was used to determine region reduces residual Fc RI binding, and substitution of proline binding of clenoliximab and inhibition by sCD4 as previously determined http://www.jimmunol.org/ for serine at residue 228 in the hinge region, stabilizes disulfide for keliximab (15). SupT1-18 cells were washed twice in PBS without bond interactions between the heavy chains of the mAb (Fig. 1). Ca2ϩ and Mg2ϩ and 2 ϫ 105 cells/well in staining buffer (PBS containing Extensive physical, kinetic, and thermodynamic comparison of 0.2% BSA and 0.1% sodium azide) were incubated with 20 ␮g/ml of sCD4 ␮ these two Abs shows no differences in their complex formation and 2 g/ml to 5 ng/ml of clenoliximab or an irrelevant IgG1 mAb (SB- 271110) for 30 min on ice. The cells were washed twice and incubated for with soluble CD4 Ag (sCD4) (2, 18). In this paper we describe the another 30 min on ice with 1/50 final dilution of secondary Ab (goat anti- in vitro functional characteristics of clenoliximab in comparison to human IgG-FITC). Cells were washed twice in cold staining buffer, and keliximab and compare the consequences of their different Fc␥R data were acquired using a Becton Dickinson (San Jose, CA) FACScan. binding properties. The results were analyzed using CellQuest software (Becton Dickinson). The epitope specificity of keliximab and clenoliximab was analyzed by guest on September 28, 2021 using SupT1-18 cells and PBMCs isolated from freshly collected whole Materials and Methods blood from healthy donors using Ficoll-Paque density gradient separation. Abs, cell lines and reagents Triplicate samples of cells at 4 ϫ 105 cells/well were treated with keliximab or clenoliximab (1.56–200 ng/ml) for 30 min on ice, followed by Fluorescent-labeled Abs used for cell binding include: OKT4 PE and another half-hour incubation with 10 ␮l each of OKT4-PE and OKT4A- OKT4A FITC (Ortho Diagnostics, Raritan, NJ); goat anti-human IgG- FITC Abs. Flow cytometric analysis of these samples was performed as FITC (Sigma, St. Louis, MO); CD3 FITC/CD19 PE, CD4 FITC/CD8 PE, above, except that the results were analyzed using Attractors software and CD14 FITC/CD16 PE (Immunotech-Coulter, Hialeah, FL); Fc receptor (Becton Dickinson). mAbs 197, AT10, and 3G8 (Medarex, Annandale, NJ); and monocyte markers anti-human CD64-FITC, CD32-FITC, and CD16-FITC (Phar- Three-way MLR and IL-2 production Mingen, San Diego, CA). Monocyte cell line THP1 and IL-2-dependent cell line CTLL-2 were obtained from the American Type Culture Collec- Human PBMCs isolated from three unrelated donor buffy coats (Red tion (Manassas, VA). Human CD4 transfected murine fibroblast cell line Cross, Philadelphia, PA) served as stimulator and responder mixes. From ϫ 5 (DAP-CD4) and human hybridoma cell line TPT10.4 were provided by Dr. each donor, 1 10 PBMCs were plated into round-bottom 96-well mi- R. P. Sekaly (McGill University, Montreal, Canada) and Dr. Sherri Mor- crotiter plate and cultured in TY medium (19) in the presence or absence of keliximab or clenoliximab (200–1000 ng/ml), in a final volume of 200 rison (University of California, Los Angeles, CA), respectively. Rheuma- ␮ toid factor (RF), one-step monocytes, and baby rabbit complement were l/well. Plates were cultured for 6 days at 37°C with 5% CO2 with addition ␮ 3 obtained from Accurate Chemical and Scientific (Westbury, NY). The re- of 1 Ci/well of [ H]thymidine/well (ICN Biomedicals, Costa Mesa, CA) during the final 8 h. IL-2 was measured by transferring 100 ␮l of 48-h-old dox indicator Alamar Blue was obtained from Biosource International ␮ (Camarillo, CA), and low IgG FBS was from HyClone (Logan, UT). Other supernatant from each well (replaced with 100 l of medium) to a round- ␮ ϫ 5 reagents such as BSA, sodium azide and Triton X-100 were purchased bottom plate containing 100 lof1 10 CTLL-2 cells cells/ml. CTLL-2 from Sigma, IFN-␥ was purchased from Boehringer Mannheim (Indianap- cultures were harvested at the end of 48 h after culturing at 37°C with 1 ␮Ci of [3H]thymidine/well for the last 7–8 h. The amount of DNA-incor- olis, IN), Ficoll-Paque from Pharmacia-LKB Biotechnology (Piscataway, 3 NJ), calcein acetomethoxy ester from Molecular Probes (Eugene, Oregon), porated [ H]thymidine from both MLR and IL-2 cultures was determined and formaldehyde from Polysciences (Warrington, PA). using a Packard (Meriden, CT) beta-scintillation counter. Mean and SEM for the triplicate cultures was calculated. Derivation and expression of clenoliximab Fc receptor-mediated adhesion assay The derivation of the modified ␥4 region and the expression of clenolix- Fc receptor binding activity of keliximab and clenoliximab were measured imab will be described elsewhere (R. Newman, K. Hariharan, M. Reff, D. as described earlier (15), except that in this assay DAP-CD4 cells express- R. Anderson, G. Braslawsky, D. Santoro, N. Hanna, P. J. Bugelski, M. ing CD4 and IFN-␥-stimulated THP1 cells were used. Fluorescence read- Brigham-Burke, C. Crysler, R. C. Gagnon, P. Dal Monte, M. L. Doyle, P. ings from triplicate samples were averaged and the SEM calculated. C. Hensley, M. P. Reddy, R. W. Sweet and A. Truneh, manuscript in preparation). Briefly, the human ␥4 region was isolated from the cell line Binding of anti-CD4 mAbs to monocytes TPT10.4 and cloned into the expression vector Anex1, which contained the variable region genes from the IgG1 mAb, keliximab followed by conver- Monocytes were isolated from fresh whole blood from healthy volunteers, sion of Ser228 and Leu235 to proline and glutamic acid, respectively, by using dextran sedimentation of RBCs followed by density separation using PCR mutagenesis (10, 15, 17). This modified IgG4 construct was subcloned one-step monocytes (20). Based on the staining by CD14 Ab, purity of the The Journal of Immunology 1927

isolated monocytes routinely exceeded 85%. Freshly isolated monocytes were plated in 96-well microtiter plates at a cell density of 4 ϫ 105/well in 200 ␮l of culture medium containing 10% heat-inactivated autologous serum and 250 U/ml IFN-␥ to induce Fc␥RI. Induction of Fc receptor expression was measured by ﬂow cytometry after 36 h using FITC-conju- gated anti-human CD64, CD32, and CD16 Abs. CD4 mAb-mediated adhesion of CD4ϩ T cells (SupT1-18) to the monocyte monolayer was determined as described in the Fc receptor-mediated adhesion assay.

Complement dependent cytotoxicity (CDC) SupT1-18 cells (5 ϫ 104) were placed in 96-well ﬂat-bottom Falcon microtiter wells containing 20% baby rabbit complement in RPMI 1640 (without phenol red and with 5% heat-inactivated, low bovine IgG FBS), CD4 mAbs (1 ␮g/ml to 0.12 ␮g/ml), and 10% alamar Blue (21). Fluores- cence was detected after 18 h at excitation/emission wavelengths of 544 nm/590 nm using Fluoroskan. Background spontaneous lysis was determined from cells grown without therapeutic agents, and maximal lysis by the addition of 0.2% Triton X-100. Percent lysis, or cytotoxicity, was calculated as: [(FL590 mn test sample Ϫ FL590 nm spontaneous lysis)/ (FL590 nm maximal lysis Ϫ FL590 nm of spontaneous lysis)] ϫ 100. Downloaded from

Ab-dependent cell-mediated cytotoxicity (ADCC) Whole blood was collected from healthy donors, and the red blood cells were lysed with ammonium chloride buffer. The remaining PBLs were FIGURE 2. Binding of clenoliximab to CD4ϩ T cells (SupT1-18 line) washed twice in HBSS and stimulated with 10 U/ml of IL-2 in RPMI 1640 and inhibition of binding by sCD4. SupT1-18 cells were incubated with containing 10% FCS for 24 h at 37°C. After 24 h, the effector PBLs were clenoliximab (2 ␮g/ml to 5 ng/ml) in the presence or absence of sCD4, as resuspended in RPMI 1640 containing 5% FCS and plated at a density of previously determined for keliximab (15). The inset shows comparative http://www.jimmunol.org/ 8 ϫ 105 cells/well. Concurrently, 1 ϫ 106 CD4ϩ SupT1-18 target cells binding of the two anti-CD4 mAbs to SupT1-18 cells. Binding of the anti- were labeled by incubating with 400 ␮Ci of 51Cr overnight at 37°C, after which they are washed four times, resuspended in RPMI 1640/5% FCS, CD4 mAbs to SupT1-18 cells is indicated by the mean ﬂuorescent intensity and added to the PBL plate at an E:T ratio of 80:1 followed by the addition (MFI) of the secondary Ab (FITC-labeled goat anti-human IgG) staining. of mAbs and then incubated for 30 min at 37°C. Wells with 100 ␮lof2% Data represent one of four experiments that yielded similar results. Triton X-100 or medium were used as maximal and spontaneous release controls, respectively. The plates were centrifuged for 3 min at 900 rpm

and incubated for4hat37°C, 5% CO2. Following incubation, the supernatant from each well was collected and the amount of radioactivity mea- Results sured on a gamma counter. Percent cell lysis was determined as: [(sample count Ϫ spontaneous release)/(maximal release Ϫ spontaneous release)] Fc-independent properties by guest on September 28, 2021 ϫ 100. Ag recognition. Although clenoliximab and keliximab share the same V regions, their different Fc domains could alter Ag recog- CD4 receptor modulation nition and complicate interpretation of their Fc-mediated activities. Thus, we examined the binding properties of these mAbs to both Receptor modulation was studied in PBMCs and in purified CD4ϩ T cells. ϩ soluble and cell surface CD4. The kinetics, energetics, and stoi- CD4 T cells were isolated from the purified PBMCs by negative selection using an Ab enrichment mixture and magnetic colloid containing dextran chiometry of binding for these mAbs to sCD4 were assessed by iron according to manufacturer’s instructions (Stemcell Technologies, Brit- BIAcore analysis, microcalorimetry, and sedimentation ultracen- ish Columbia, Canada). Cells (1 ϫ 106/ml) were cultured with clenolix- trifugation, which have been already described (18). Briefly, both imab or keliximab (100 ng/ml) for 24 h in the presence or absence of IFN-␥ mAbs showed little aggregation alone or in complex with sCD4 (313 U/ml). The cells were washed twice and dual stained with OKT4 PE Ϯ and CD3 FITC for measurement of CD4 and CD3 expression, respectively, and showed equivalent binding to sCD4 (1.92 0.05 sCD4/mAb), and with OKT4-PE and OKT4A-FITC for measuring epitope masking by indicating similar Ag binding characteristics and high functional keliximab and clenoliximab. The percentage of T cells (and CD4/CD8 activity of the mAb preparations (18). ratio), B cells, and monocytes in the PBMC preparations was determined The binding of these mAbs to cell surface CD4 was examined by dual staining with CD3-FITC/CD19-PE, CD4-FITC/CD8-PE, CD14- on the thymocyte cell line SupT1 (subclone 18) and on PBMCs. FITC/CD16-PE Abs, respectively. Isotype controls were included in all assays. Data were acquired by flow cytometry as described above for the Fig. 2 shows saturable binding of keliximab and clenoliximab to ␮ cell binding assays. SupT-18 cells with equivalent ED50 of 0.3 g/ml. Binding of In blocking experiments with Fc receptor mAbs, PBMCs were cultured clenoliximab to these CD4ϩ T cells was completely inhibited by for 4 h with keliximab (50 ng/ml) in the presence or absence of 5 ␮g/ml of excess sCD4, as previously shown for keliximab (15). No binding Fc receptor mAbs: 197 (anti-FcRI), AT10 (anti-FcRII), and 3G8 (anti- FcRIII) and analyzed for CD4 receptor modulation as described previously. was detected for the irrelevant, humanized, mAb SB-271110. For CD4ϩ T cell and monocyte coculture assays, freshly isolated mono- To allow direct comparison of the two human CD4 mAbs, a cytes (5 ϫ 105 cells/well) were plated in 48-well tissue culture plates and competition assay was established using the CD4 mAbs OKT4A cultured for 24 h in 1 ml of RPMI 1640 containing 10% heat inactivated and OKT4. We previously showed that the epitope recognized by ␥ ϩ autologous serum and 313 U/ml of IFN- . CD4 T cells isolated from the keliximab resides within domain 1 of CD4 and overlaps with that same donor the next day (as described above) were added to the monocyte cultures at a CD4ϩ T cell:monocyte ratio of 4:1 and cultured for another of OKT4A but not OKT4, which binds in the 3/4 extracellular 24 h in the presence or absence of 100 ng/ml of clenoliximab or keliximab. domains of CD4 (15). Based on this recognition, keliximab and The cells were recovered and stained for CD4 receptor and analyzed by clenoliximab will block the binding of OKT4A but not OKT4. flow cytometry as mentioned earlier in the cell binding experiment. Both mAbs showed comparable competition of OKT4A binding To determine the effect of RF, PBMCs were cultured with clenoliximab or keliximab (100 ng/ml) in the presence or absence of RF (6 U/ml) for and did not affect the binding of OKT4 (Fig. 3, A and B). On 24 h. Cells were then recovered and stained for CD4 receptor and analyzed SupT1-18 cells, the ED50 values for keliximab and clenoliximab by flow cytometry. were 55 ng/ml and 65 ng/ml, respectively, and on PBMCs the 1928 ELIMINATION OF Fc RECEPTOR FUNCTIONS OF A CD4 mAb

FIGURE 3. Saturable binding of keliximab and clenoliximab analyzed in the CD4ϩ T cell line, SupT1-18 (A) and in human PBMCs (B). Both SupT1-18 cells and PBMCs were treated with varying concentrations of keliximab or clenoliximab (200 ng/ml to 1.56 ng/ml), followed by staining with OKT4A Ab (interferes with anti-CD4 mAb binding to domain 1 of CD4 receptor) and OKT4 Ab (noninterfering, as it binds to domain 4). Staining with both OKT4A Downloaded from and OKT4 Abs was measured by flow cytometry and MFI values were calculated using Attractors software (Becton Dickinson). Saturable binding was identified for each mAb, as the concentration at which the MFI of OKT4A and OKT4 Abs reached a plateau. Results are expressed as the mean Ϯ SEM of triplicate samples. http://www.jimmunol.org/ ED50 for both mAbs was 50 ng/ml. We conclude that the different (CD16) receptors occurs through determinants within the heavy Fc domains of clenoliximab and keliximab do not affect their rec- chain constant regions. ognition of CD4 in solution or on the cell surface. To assess Fc receptor binding activity, an assay was developed Inhibition of IL-2 production and proliferative responses in three that measured the capacity of CD4 mAbs to mediate the attach- way MLR. Most CD4 mAbs inhibit MLR, which measures al- ment of Fc receptor expressing THP1 cells to the adherent CD4ϩ loantigen-driven T cell proliferation and IL-2 production (15, 22). fibroblasts (bridging assay). This assay measures the CD4 and Fc ϩ The MLR is dependent on the presence of CD4 T cells, and much receptor binding functions of mAbs simultaneously, and thus may of this requirement stems from the interaction of the CD4 receptor be an in vitro correlate of Fc receptor and mAb-mediated depletion with MHC class II molecules on APC. Although the mechanism is ϩ ␥

of CD4 cells in vivo. IFN- -stimulated THP1 cells express a high by guest on September 28, 2021 not well defined, mAbs specific for domain 1 of CD4 appear to act level of the high affinity receptor Fc␥RI and modest levels of the through blockade of the interaction of CD4 with MHC class II low affinity receptors Fc␥RII and Fc␥RIII. Keliximab facilitated molecules. In a three-way MLR, keliximab and clenoliximab were adhesion of the nonadherent THP1 cells to the adherent CD4ϩ equivalent in their ability to inhibit the proliferative response of T fibroblasts, which are dependent on the Fc domain of keliximab, cells and IL-2 production (Table I). Thus, the different Fc domains Ј because its F(ab )2 fragment was not active (Fig. 4A). Adhesion on these mAbs did not affect their ability to block CD4-dependent also requires the Ag recognition site of keliximab, because it was T cell responses in vitro. inhibited by sCD4 (Fig. 4A). In contrast, clenoliximab showed no activity in this assay (Fig. 4B). Because the two mAbs bind equiv- Fc-dependent properties of keliximab and clenoliximab alently to CD4, the lack of activity with clenoliximab must reflect Binding of CD4 mAbs to Fc receptors. The ability of a CD4 the absence of binding to Fc receptors (Fig. 4B). mAb to bind to human Fc receptors on monocytes, macrophages, A second assay investigated the ability of the CD4 mAbs to granulocytes, and NK cells is an important in vitro indicator of mediate adhesion of IFN-␥ stimulated human monocytes (express- immune effector capability related to in vivo mechanisms of CD4 ing IgG Fc receptors) and the human T cell line SupT1–18 (Fig. receptor modulation and cell depletion. Binding to the high affinity 5A). Flow cytometric analysis of these monocytes showed strong ␥ ␥ ␥ Fc RI (CD64) and the low affinity Fc RII (CD32) and Fc RIII up-regulation of CD64 (Fc␥RI) expression (Fig. 5B). The moder- ate level of CD32 (Fc␥RII) and low level of CD16 (Fc␥RIII) expression on nonactivated monocytes remain unaffected by IFN-␥ a Table I. Effect of anti-CD4 mAbs on three-way MLR (IC50) treatment. As expected, keliximab showed good binding activity ␮ with an IC50 of 0.03 g/ml (Fig. 5A). Although donor-to-donor Mean Proliferation Mean IL-2 Production differences were observed, clenoliximab was 10- to Ͼ100-fold less (IC50) ng/ml (IC50) ng/ml active than keliximab, consistent with a reduced Fc receptor bind- Clenoliximab 14.6 Ϯ 2.7 3 Ϯ 0.2 ing activity for its modified IgG4 region. Keliximab 11.5 Ϯ 1.4 4 Ϯ 0.3 CDC and ADCC effector properties of keliximab and clenolix- Ͼ Ͼ OKT8 1000 1000 imab. To assess complement mediated activity, the cytotoxic a Freshly isolated human PBMCs from three separate unrelated blood samples ability of test and control mAbs was evaluated in the presence and were cultured with keliximab or clenoliximab at varying concentrations from 1 ␮g/ml absence of baby rabbit complement using the SupT1-18 cell line as to 200 ng/ml. Proliferative response of T cells was determined by measuring the [3H]thymidine incorporation during the last8hofa6-day culture. Effect of anti-CD4 a target. Detection of cytotoxicity, measured as lack of fluores- mAbs on IL-2 production was determined in 48-h supernatents by measuring prolif- cence, was noted after an 18-h incubation using the metabolic ac- eration of IL-2-dependent cell line CTLL-2. Concentration of mAbs at which 50% tivity indicator, alamar Blue. Neither clenoliximab nor keliximab inhibition of proliferation or IL-2 production (IC50) occurred was calculated from the mean Ϯ SEM of triplicate cultures and is represented above. (Fig. 6A) mediated cell lysis in the presence of complement. The The Journal of Immunology 1929

FIGURE 4. Fc receptor binding characteristics of keliximab and clenoliximab. A, Keliximab-mediated adhesion of IFN-␥-treated monocytic cell line (THP1) to CD4ϩ fibroblast transfectants. Also shown is the inhibition of this CD4 mAb-mediated adhesion by sCD4 along with the effect of truncated form of keliximab. B, Comparison of IgG1 and IgG4 anti-CD4 mAbs in FcR-CD4-mediated cell-cell adhesion assay. Percent adhesion of THP-1 cells to CD4ϩ T cells was calculated as: (fluorescence of test samples/total fluorescence) ϫ 100. Data points are the mean Ϯ SEM of triplicate cultures. Downloaded from lack of activity of keliximab is consistent with our previous ob- sessed in freshly isolated PBMCs from healthy donors. Keliximab servation of its poor binding to C1q and inability to fix human significantly reduced the intensity of staining with OKT4 mAb complement (14). OKT4, a murine Ig2b mAb, was used in this relative to the untreated control (Figs. 7 and 8A). Keliximab also

assay as a positive control mAb and showed cytotoxicity at con- induced comodulation of CD3, although to a lesser extent, as http://www.jimmunol.org/ centrations ranging from 1 ␮g/ml to 0.012 ␮g/ml, with lysis as shown by the reduction of CD3 staining intensity (Figs. 7 and 8B). high as 58% of total cells. None of the mAbs were active in the In contrast, modulation of CD4 or CD3 was not observed with absence of complement. Thus, neither keliximab nor clenoliximab clenoliximab. This difference in modulation between keliximab promote cell lysis through complement effector mechanisms. and clenoliximab was not due to the extent of Ag coating, because ADCC is a mechanism by which Fc receptor-positive effector they bound equivalently to these cells, as previously shown in Fig. cells can lyse target cells that have adherent Ag-specific Ab. Serial 3B. The keliximab-induced comodulation of CD3 was specific for dilutions of the CD4 mAbs were incubated with 51Cr-labeled CD4ϩ cells, because there was no change in CD3 intensity in the SupT1-18 cells, followed by addition of IL-2-stimulated PBLs as CD4Ϫ/CD3ϩ population (primarily CD8ϩ T cells; Fig. 8B). The effector cells. The results in Fig. 6B show keliximab-mediated spe- surface modulation of CD4 and CD3 on CD4ϩ cells was selective by guest on September 28, 2021 cific cell lysis as previously observed (15). In contrast, clenolix- because CD25 and CD71, markers for early T cell activation, were imab did not induce lysis above background. not changed (data not shown). CD4 receptor modulation. CD4 mAbs can induce modulation of Addition of IFN-␥ to PBMC cultures, which causes Fc␥RI up- CD4 on the surface of T cells, most likely through receptor inter- regulation on monocytes, significantly accelerated CD4 and CD3 nalization and/or receptor stripping (23, 24). In vitro, this loss of modulation by keliximab and resulted in almost complete loss of CD4 appears to require the presence of monocytes. The effect of CD4 receptors from the cell surface (Fig. 9A). Moreover, under keliximab and clenoliximab on CD4 receptor modulation was as- these conditions, a 40% reduction in CD4 was observed with

FIGURE 5. A, Keliximab and clenoliximab-mediated adhesion of CD4ϩ T cells to IFN-␥-induced Fc receptor-expressing monocytes. Anti-CD4 mAbs serve as a bridge between the monocytes on the microtiter plates and fluorescent dye loaded SupT1-18 T cells. Percent binding of SupT1–18 cells to monocytes was calculated as in Fig. 4B (fluorescence of test samples/total fluorescence) ϫ 100. Data represent one of three experiments that showed identical results (15). B, Freshly isolated human monocytes were cultured for 36 h with 250 U/ml human IFN-␥, after which induction of Fc receptor expression was measured by staining with anti-human CD64-FITC, CD32-FITC, and CD16-FITC Abs. Flow cytometric analysis was performed using CellQuest software (Becton Dickinson). 1930 ELIMINATION OF Fc RECEPTOR FUNCTIONS OF A CD4 mAb

FIGURE 6. C1q binding properties of keliximab and clenoliximab. A, CDC of anti-CD4 mAbs. Evaluation of CDC of anti-CD4 mAbs was performed in the presence and absence of baby rabbit complement using the SupT1-18 CD4ϩ T cell line as target. Cytotoxicity was measured as lack of ﬂuorescence

after incubation for 18 h and detected using the metabolic activity indicator, alamar Blue. Mean spontaneous lysis of cells (measured in the absence of Downloaded from anti-CD4 mAbs) was 3940 Ϯ 150 ﬂuorescence units (F.U.) and mean maximal lysis was 456 Ϯ 6 F.U. in the presence of 0.2% Triton X-100. B, Ab-dependent cellular cytotoxicity was analyzed for both keliximab and clenoliximab using the classical method of cell-mediated cytolysis of chromium labeled CD4ϩ SupT1-18 cells. Effector cells are human PBLs from a Red Cross buffy coat. The results are expressed as the mean Ϯ SD of percent cytotoxicity.

clenoliximab (Fig. 9A). Cell fractionation studies showed that this lower modulation activity of clenoliximab is consistent with its http://www.jimmunol.org/ modulation is induced by monocytes. Purified monocytes were reduced affinity for this receptor. incubated with or without IFN-␥ and then added to CD4ϩ T cells RF could contribute to Ab-mediated modulation of CD4 in vivo. from the same donor. As observed in the PBMC cultures, partial RFs are aberrant Igs often observed in patients with RA. These modulation was observed with untreated monocytes and IFN-␥ molecules bind to human Ab and can lead to the formation of treatment resulted in almost complete CD4 modulation with kelix- immune complexes (26). Purified RF was tested for its effect on imab and about 30% reduction with clenoliximab (Fig. 9B), indi- CD4 mAb-induced modulation in PBMCs. RF enhanced modula- cating that monocytes are required for CD4 mAb-induced modu- tion by keliximab, but its effect was most noticeable with cleno- lation. Subsequently, the necessity of Fc receptor-expressing cells liximab (Fig. 11). RF enhancement of CD4 mAb modulation may to trigger modulation of CD4 receptor was examined. Purified result directly from cross-linking of cell surface CD4 or from sub- by guest on September 28, 2021 CD4ϩ T cells alone show little modulation with keliximab or sequent recognition of the complex by Fc receptors. clenoliximab (Fig. 9C). IFN-␥ treatment of the purified CD4ϩ T cells resulted in minimal modulation, perhaps due to the presence of residual Fc receptor expressing cells, such as monocytes or NK Discussion cells. Because the enhancement of modulation by IFN-␥, strongly Given the critical involvement of T cells in the development and implicated Fc receptor involvement, blocking mAbs to the Fc re- maintenance of autoimmune diseases, immunosuppression by CD4 ceptors were added to PBMC cultures containing either keliximab mAb therapy has become an important strategy for the treatment of or clenoliximab. Keliximab-induced modulation was partially re- these diseases. CD4 mAbs can mediate in vivo immunomodulatory versed by mAb 197, which is reported to partially inhibit Ab bind- effects through 1) removal of CD4ϩ T cells by apoptosis or comple- ing to Fc␥RI/CD64 (Fig. 10) (25). Little, if any, effect was ob- ment-mediated lysis of mAb-coated CD4ϩ T cells; 2) receptor mod- served with either mAb AT10 or mAb 3G8, directed against ulation caused by internalization or stripping of the CD4 receptor human Fc␥RII and Fc␥RIII (monocyte isoform), respectively. As from the cell surface; 3) inhibition of T cell activation by antagonism expected, clenoliximab did not induce modulation on these un- of CD4-MHC class II interactions, possibly leading to induction of stimulated PBMCs, and there was no effect with addition of the Fc anergy; and 4) “negative” signaling via the CD4 receptor (7, 27, 28). receptor mAbs. We conclude that monocyte-mediated CD4 mod- We have investigated the first three mechanisms in our in vitro func- ulation by the CD4 mAbs is primarily dependent on Fc␥RI. The tional characterization of clenoliximab and keliximab. Although both

FIGURE 7. Keliximab and clenoliximab induced CD4 and CD3 receptor modulation. Shown are flow cytometric two-dimensional scatter plots of human PBMCs incubated with 100 ng/ml of keliximab or clenoliximab or medium without added Ab and dual stained for CD4 (OKT4) and CD3 markers after 24 h of culture. The Journal of Immunology 1931 Downloaded from FIGURE 8. CD4 (A) and CD3 (B) receptor modulation in PBMCs. Freshly isolated human PBMCs were cultured with 100 ng/ml of keliximab or clenoliximab or medium, and the effect on CD4 and CD3 surface markers was measured by flow cytometry after 24 h of culture. MFI of CD4 and CD3 markers were determined using Attractors software (Becton Dickin- son), and the mean of triplicate cultures are represented above. http://www.jimmunol.org/ mAbs can act through these mechanisms, the Fc-dependent activities of T cell depletion and receptor modulation was strongly reduced for clenoliximab. Fc receptors expressed on human peripheral blood cells play an important role in triggering a variety of cytotoxic, phagocytic, and inflammatory functions (25, 29, 30). The site for binding of IgGs FIGURE 9. Effect of IFN-␥ on CD4 receptor modulation. A and C, ␥ ϩ by guest on September 28, 2021 to Fc RI is proposed to be Leu-Leu-Gly-Gly-Pro-Ser (Kabat po- Modulation in PBMCs or purified CD4 T cells. These cells were freshly sition numbers 247–252), which lies at the border of the hinge and isolated from a healthy volunteer and cultured separately in the presence or CH2 regions (31). Human IgG4 and mouse IgG2b, which have low absence of IFN-␥ and with or without keliximab or clenoliximab. B, CD4 affinity for Fc␥RI, contain the sequence Phe-Leu-Gly-Gly-Pro-Ser receptor modulation in cocultures. Monocytes were freshly isolated from and Leu-Glu-Gly-Gly-Gly-Pro-Ser, respectively, at the Fc␥RI the same donor a day earlier and cultured in IFN-␥-containing medium. ϩ binding site. Based on this observation and other mutation and The following day some of the isolated CD4 T cells were added to the reverse substitution studies using mouse IgG2b (32), human IgG3 monocyte cultures at a ratio of (4:1) and cultured in the presence or absence (33), or IgG4 (34), Leu248 was mutated to Glu in the IgG4 version of 100 ng/ml of keliximab or clenoliximab. All the cells were recovered after 24 h and analyzed for CD4 receptor density by flow cytometry. Data of keliximab to yield a mAb with a predicted affinity for Fc␥RI to 241 represent one of two experiments that indicated similar results with differ- be 1000-fold lower than the IgG1 mAb. Further, Ser was re- ent donors. placed by Pro to enhance stability of the IgG4 heavy chain dimer (35), thus producing clenoliximab. The two CD4 mAbs were compared for activity mediated by the Cells with cytotoxic potential that bind mAbs via Fc receptor Fab regions (cell surface binding and MLR) and the Fc domains can mediate ADCC directed to Ab-coated target cells (29). Com- (Fc receptor binding, ADCC, and CDC). As expected, clenolix- parison of ADCC effector properties of clenoliximab and keliximab and keliximab bind to the same epitope on CD4 with equiv- imab indicated that, unlike the IgG1 mAb (15), IgG4 mAb did not alent affinity and specificity. Also, these mAbs show equivalent mediate specific lysis of CD4ϩ cells. These, in addition to our in interference with T cell activation, as measured by inhibition of vivo studies (M. P. Reddy and A. Truneh, unpublished observa- proliferation and IL-2 production by T cells in primary MLR. Sim- tions) contrast with observations for an IgG4 mAb directed to the ilar results were obtained with an IgG4 construct lacking the hinge T cell surface molecule, CD52 (Campath-1), which mediated Fc and CH2 region mutations (unpublished observations), indicating effector functions and caused T cell depletion in vivo (36). These that substitutions in these domains of clenoliximab do not affect its differences may be due to different Ag targets. However, as noted ability to block CD4-dependent T cell responses in vitro. above, further alterations of IgG4 were required to eliminate Fc As anticipated, the modified IgG4 CD4 mAb, clenoliximab, ex- effector functions. Such constructs retain additional desirable at- hibited markedly reduced Fc receptor binding activities, when tributes such as prolonged inherent circulating half life in vivo and compared with keliximab, for binding to cell surface CD4 on trans- effective suppression of T-dependent responses (M. P. Reddy and fected fibroblasts and to Fc receptor on IFN-␥-induced THP1 cells A. Truneh, unpublished observations). and freshly isolated monocytes. The Leu248 mutation to Glu was The complement system contains, among its various functional essential for elimination of Fc receptor binding because other IgG4 components, the ability to interact with certain types of Abs in a constructs lacking this substitution were indistinguishable from the manner which leads to cell lysis and destruction. Human IgG1 Abs IgG1 mAb in this assay (data not shown). normally possess the capability to bind C1q and deplete target cells 1932 ELIMINATION OF Fc RECEPTOR FUNCTIONS OF A CD4 mAb

and in the clinic (J. Aldrich, F. Breedveld, C. Davis, M. Elliott, M. Jackson, E. Keystone, R. Levy, J. Sany, J. Tesser, A. Truneh, M. Weisman, C. Wiesenhutter, D. Yocum, J. Zhu and U. Mason, manuscript in preparation). Possible explanations for this difference in vivo include: 1) up-regulation of Fc receptors under inflammatory in vivo disease conditions, 2) higher Fc receptor levels on tissue resident macrophages than on nonstimulated circulating blood monocytes, and 3) the presence of factors such as RF that can cross-link Abs bound on cell surfaces. Consistent with these possibilities, clenoliximab induced partial CD4 receptor modulation in vitro in the presence of RF, and under stimulatory conditions that up-regulated Fc␥RI expression on monocytes apparently resulting in weak cross-linking of clenoliximab on CD4 cells, leading to partial down modulation of CD4. ␥ FIGURE 10. Effect of Fc R mAbs on CD4 modulation in PBMCs. Partial or inappropriate signaling of T cells also results in the Freshly isolated PBMCs were cultured with or without keliximab or cleno- triggering of apoptotic pathways leading to apoptotic cell death. liximab in the presence of absence of the indicated Fc receptor Abs for 4 h. CD4 receptor modulation was then analyzed by flow cytometry. Results are Another attractive characteristic of the modified IgG4 mAb, cleno- expressed as mean Ϯ SEM of triplicate cultures. liximab, is its lack of apoptotic activity unlike the original IgG1

keliximab, demonstrated in vitro using freshly isolated PBMCs (J. Downloaded from Fishman-Lobell, P. Tsui, M. Reddy, R. DiPrinzio, R. Sweet, A. bearing surface Ags for which they have speciﬁcity. Other human Truneh and C. Eichman, manuscript in preparation). isotypes such as IgG4 exhibit reduced ability to bind C1q and thus Based on the observations from this study, clenoliximab func- would be unable to deplete target cells (37). Both keliximab and tions as classical receptor antagonist, through blockade of the in- clenoliximab were ineffective in promoting cell lysis through teraction of CD4 with its counterreceptor MHC class II, and mod-

complement effector mechanisms. It was noted earlier that ulation of CD4 from the cell surface. T cells that drive the http://www.jimmunol.org/ keliximab binds C1q poorly and thus is unable to fix human autoimmune response are believed to be weakly self-reactive cells complement (15). that have escaped the normal mechanisms of peripheral tolerance. The benefit of the altered Fc domains of clenoliximab was fur- These cells depend on the participation of coreceptors, such as ther evident in CD4 receptor modulation. The ability of CD4 mAbs CD4, for the full elaboration of an immune response. Blocking the to modulate the CD4 receptors from the resting T cells appears to coreceptors would deprive these T cells of crucial cosignaling be induced by Fc receptor-mediated cross-linking which is re- functions, resulting in partial activation, or anergy. In the mouse ported not to be required in activated T cells (38). Three sets of this approach appears sufficient and preferable to depletion of CD4 experimental evidence demonstrate that CD4 receptor modulation cells that can reduce efficiency and lead to long-term suppression by keliximab was dependent on the presence of Fc receptor-ex- of these cells (40). Indeed in HuCD4 Tg mice and in chimpanzee, by guest on September 28, 2021 ϩ pressing cells: 1) purified CD4 T cells ablated modulation nor- there is modulation without depletion. Treatment with clenolix- mally observed with keliximab in unfractionated PBMC cultures; imab may allow the immune system to regulate reactivity to 2) conditions which cause Fc␥RI up-regulation (in the presence of self-Ag resulting in long-lasting effects in chronic autoimmune dis- IFN-␥), caused complete loss of CD4 from the cell surface; and 3) eases without the often serious side effects observed with broad modulation induced by keliximab was partially reversed by mAb immunosuppressive agents (41). 197, which blocks binding to Fc␥RI. In addition, keliximab, but Other factors that can potentially influence the in vitro and in not clenoliximab, was also found to induce TNF-␣ and IL-6 pro- vivo properties of mAbs to cell surface molecules include the na- duction by CD4 mAb-treated PBMCs, another effect mediated by ture of the targeted molecule, its cellular and tissue distribution, Fc receptor cross-linking (data not shown) (36, 39). and the Ag density on the cell surface. Thus, a judicious selection The reduced ability of clenoliximab to cause CD4 receptor mod- of the target as well as appropriate modification of the effector ulation under these in vitro conditions is consistent with its weak function of the selected mAb can have considerable impact on the binding to Fc receptors, which was one of the deliberate design pharmacological outcome of the use of these mAbs in vivo. features of this mAb. However, clenoliximab causes strong CD4 receptor modulation in animal models (HuCD4 transgenic mice Acknowledgments (40) and chimpanzees, Newman et al., manuscript in preparation), We thank Christopher Eichman and Rocco DiPrinzio for flow cytometry support, and John Baldoni, Michael Elliott, John Erickson, Marcia Fed- erici, and Anthony Lubiniecki for many helpful discussions.

References 1. Sweet, R. W., A. Truneh, and W. A. Hendrickson. 1991. CD4: its structure, role in immune function and AIDS pathogenesis, and potential as a pharmacological target. Curr. Opin. Biotechnol. 2:622. 2. Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J. Arthos, J. Rosenberg, P. J. Maddon, R. Axel, and R. W. Sweet. 1988. A soluble of CD4 (T4) protein inhibits AIDS virus infection. Nature 331:82. FIGURE 11. Effect of RF on modulation of CD4 receptors on T cells by 3. Panayi, G. S., J. S. Lanchbury, and G. H. Kingsley. 1992. The importance of the keliximab or clenoliximab. PBMCs were cultured with keliximab or cleno- T cell in initiating and maintaining the chronic synovitis of rheumatoid arthritis. liximab (100 ng/ml) or medium without any added Ab in the presence or Arthritis Rheum. 35:729. 4. Waldmann, H., and S. Cobbold. 1993. The use of monoclonal antibodies to absence of puriﬁed RF at 6 U/ml for 24 h. CD4 receptor density was achieve immunological tolerance. Immunol. Today 14:245. measured by ﬂow cytometry by dual staining the cultured cells with OKT4 5. Bach, J. F. 1993. Immunosuppressive therapy of autoimmune diseases. Immunol. and OKT4A Abs. Data represent the mean Ϯ SEM of triplicate cultures. Today 14:322. The Journal of Immunology 1933

6. Herzog, C., C. Walker, M. Wolfgang, P. Rieber, C. Reiter, G. Riethmuller, 24. Brett, S. J., W. Rowan, M. Smith, M. Batholomew, and J. P. Tite. 1997. Differ- P. Wassmer, H. Stockinger, O. Madic, and W. J. Pichler. 1989. Anti-CD4 anti- ential functional effects of a humanized anti-CD4 antibody on resting and acti- body treatment of patients with rhuematoid arthritis. I. Effect on clinical course vated human T cells. Immunology 91:346. and circulating T cells. J. Autoimmun. 2:627. 25. Deo, Y. M., R. F. Graziano, R. Repp, and J. G. J. vande Winkel. 1997. Clinical 7. Hafler D. A., J. Ritz, S. Schlossman, and H. Weiner. 1988. Anti-CD4 and anti- significance of IgG Fc receptors and Fc␥R-directed immunotherapies. Immunol. CD2 monoclonal antibody infusions in subjects with multiple sclerosis: immu- Today 18:127. nosuppressive effects and human anti-mouse responses. J. Immunol. 141:131. 26. Nordstrom E, E. Moller, and M. Abedi-Vaugerdi. 1998. Induction of IgG rheu- 8. Mathieson, P.W., S. P. Cobbold, and P. Hale. 1990. Monoclonal antibody therapy matoid factor (RF) production by antibody-antibody (RF-like) immune complex- in systemic vasculitis. N. Engl. Med. 323:250. es: the role of T cells, complement and Fc␥ receptors. J Autoimmun. 11:131. 9. Morel P., C. Vincent, G. Cordier, G. Panaye, E. Carosella, and J. P. Revillard. 27. Walker, C., C. Herzog, P. Rieber, G. Riethmuller, W. Muller, and W. J. Pichler, 1990. Anti-CD4 monoclonal antibody administration in renal transplanted pa- 1989. Anti-CD4 antibody treatment of patients with rheumatoid arthritis. II. Ef- tients. Clin. Immunol. Immunopathol. 56:311. fect of in vivo treatment on in vitro proliferative response of CD4 cells. 10. Newman R., J. Alberts, and D. Anderson. 1992. “Primatization” of recombinant J. Autoimmun. 2:643. antibodies for immunotherapy of human diseases: a macaque/human chimeric 28. Mannie, M. D., S. K. Rendall, P. V. Arnold, J. P. Nardella, and G. A. White. antibody against human CD4. Bio/Technology 10:1455. 1996. Anergy-associated T cell antigen presentation: a mechanism of infectious 11. Van der Lubbe, P.A., C. Reiter, F. C. Breedveld, K. Druger, M. Schattenkirchner, tolerance in experimental autoimmune encephalomyelitis. J. Immunol. 157:1062. M. E. Sanders, and G. Riethmuller. 1993. Chimeric CD4 monoclonal antibody 29. Fanger, M. W., L. Shen, R. F. Graziano, P. M. Guyre. 1989. Cytotoxicity me- cM-T412 as a therapeutic approach to rheumatoid arthritis. Arthritis Rheumatism diated by human Fc receptor for IgG. Immuol. Today 10:92. 36: 1375. 30. Guyre, P. M., R. F. Graziano, B. A. Vance, P. M. Morganelli, and M. W. Fanger. 12. Carteron, N. L., D. Wofsy, and W. E. Seaman. 1988. Treatment of murine lupus 1989. Monoclonal antibodies that bind to distinct epitopes on Fc␥RI are able to Ј with F(ab )2 fragment of monoclonal antibody to L3T4. J. Immunol. 142:1470. trigger receptor function. J. Immunol. 143:1650. 13. Carteron, N. L., D. Wofsy, and W. E. Seaman. 1988. Induction of immune tol- 31. Burton, R., and J. M. Woof. Human effector functions. 1992. Adv. Immunol. 51:1. erance during administration of monoclonal antibody to L3T4 does not depend on 32. Duncan, A. R., J. M. Woof, L. J. Patridge, and G. Winter. 1988. Localization of depletion of L3T4ϩ cells. J. Immunol. 140:713. the binding site for the high-affinity Fc receptor on IgG. Nature 332:563. 14. Hutchings, P., L. O’Reilly, N. M. Parish, H. Waldman, and A. Cooke. 1992. The

33. Lund, J., G. Winter, P. T. Jones, P. T. Pound, M. R. Walker, P. J. Artymiuk, Downloaded from use of a non-depleting anti-CD4 monoclonal antibody to re-establish tolerance to Y. Arta, D. R. Burton, R. Jefferies, and J. M. Woof. 1991. Human Fc␥RII interact cells in NOD mice. Eur. J. Immunol. 22:1913. with distinct but overlapping sites on human IgG. J. Immunol. 147:2657. 15. Anderson, D., K. Chambers, N. Hanna, J. Leonard, M. Reff, R. Newman, J. Baldoni, D. Dunleavy, M. Reddy, R. Sweet, and A. Truneh. 1997. A primatized 34. Alegre, M.-L., A. M. Collins, V. L. Pulito, R. A. Brosius, W. C. Olson, MAb to human CD4 causes receptor modulation, without marked reduction in R. A. Zivin, R. Knowles, J. R. Thislethwaite, L. K. Jolliffe, and J. A. Bluestone. CD4ϩ T cells in chimpanzees: in vitro and in vivo characterization of a MAb 1992. Effect of single amino acid mutation on the activating and immunosup- (IDEC-CE9. 1) to human CD4. Clin. Immunol. Immunopathol. 84:73. pressive properties of a humanized OKT3 monoclonal antibody. J. Immunol. 16. Kon, O. M., B. S. Sihra, C. H. Compton, T. B. Leonard, A. B. Kay and N. C. Barnes. 148:3461. 35. Angal, S., D. J King, M. W. Bodmer, A. Turner, A. D. G. Lawson, G. Roberts,

1998. Randomized, dose-ranging, placebo-controlled study of chimeric antibody to http://www.jimmunol.org/ CD4 (keliximab) in chronic severe asthma. Lancet 352:1109. B. Pedle, and J. R. Adair. 1993. A single amino acid substitution abolishes the 17. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. 1991. heterogeneity of chimeric mouse/human (IgG4) antibody. Mol. Immunol. 30:105. Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health 36. Issacs, J. D., M. G. Wing, D. J. Greenwood, L. B. Hazelman, G. Hale, and and Human Services, Bethesda, MD, NIH Publication no. 91-3242. H. Waldmann. 1996. A therapeutic human IgG4 monoclonal antibody that de- 18. Doyle, M. L., M. Brigham-Burke, M. N. Blackburn, I. S. Brooks, T. M. Smith, pletes target cells in humans. Clin. Exp. Immunol. 106:427. R. Newman, M. Reff, W. F. Stafford III, R. W. Sweet, A. Truneh, et al. 1999. 37. Garred, P., T. E. Michaelson, and A. Aase. 1989. The IgG subclass pattern of Measurement of protein interaction bioenergetics: application to structural vari- complement activation depends upon density and antibody and complement ac- ants of an anti-sCD4 antibody. Methods Enzymol., In press. tivation. Scand. J. Immunol. 30:379. 19. Jonak, L. Z., E. Henri, S. Trulli, C. P. Jensen, and K. A. Muirhead. 1992. Ma- 38. Bartholomew, M., S. Brett, K. Barber, C. Rossman, S. Crowe, and J. Tite. 1995. nipulation of human B cells to confer immortality. Hum. Antibody Hybridomas Functional analysis of the effects of a fully humanized anti-CD4 antibody on 3:177. resting and activating human T cells. Immunology 85:41.

20. Boyum, A. 1983. Isolation of human blood monocytes with Nycodenz, a new 39. Bolt, S., E. Routledge, I. Lloyd, L. Chatenoud, H. Pope, S. D. Gorman, M. Clark, by guest on September 28, 2021 non-ionic iodinated gradient medium. Scand. J. Immunol. 17:429. and H. Waldmann. 1993. The generation of a humanized, non-mitogenic CD3 21. Gazzano, S., H. P. Ralph, T. C. Ryskamp, A. B. Chen, and V. R. Mukku. 1996. monoclonal antibody which retains in vitro immunosuppressive properties. Eur. A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 J. Immunol. 23:403. monoclonal antibody. J. Immunol. Methods 202:163. 40. Podolin, P. L., E. F. Webb, M. Reddy, and D. E. Griswold. 1999. Inhibition of ϩ 22. Pulito, V. L., V. A. Roberts, and J. R. Adair. 1996. Humanization and molecular contact sensitivity in human CD4 transgenic mice by human CD4-speciﬁc ϩ modeling of the anti-CD4 monoclonal antibody, OKT4A. J. Immunol. 156:2840. monoclonal antibodies: CD4 T cell depletion is not required. Immunology, In 23. Horneff, G., F. Emmrich, and G. R. Burmester. 1993. Advances in immunother- press. apy of rheumatoid arthritis: clinical and immunological ﬁndings following treat- 41. Choy, E. H. S., G. H. Kingsley, and G. S. Panayi. 1998. Monoclonal antibody ment with anti-CD4 antibodies. Br. J. Rheumatol. 32:39. therapy in rheumatoid arthritis. Br. J. Rheumatol. 37:484.