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Clinical ImmunologyVol. 98, No. 2, February, pp. 164–174, 2001doi:10.1006/clim.2000.4975, available online at http://www.idealibrary.com on

Modification of the Fc Region of a Primatized IgG Antibody to HumanCD4 Retains Its Ability to Modulate CD4 Receptors but Does Not

Deplete CD41 T Cells in Chimpanzees

Roland Newman,*,1 Kandasamy Hariharan,* Mitchell Reff,* Darrel R. Anderson,* Gary Braslawsky,*Denise Santoro,* Nabil Hanna,* Peter J. Bugelski,† Michael Brigham-Burke,† Carl Crysler,†

Robert C. Gagnon,† Paul Dal Monte,† Michael L. Doyle,† Preston C. Hensley,† Manjula P. Reddy,†Raymond W. Sweet,† and Alemseged Truneh†

*IDEC Pharmaceuticals Corporation, 11011 Torreyana Road, San Diego, California 92121;

and †SmithKline-Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Keliximab, a Primatized IgG1 CD4 mAb, was recon-figured to an IgG4 antibody. The g4 constant regionwas further modified by substituting glutamic acid forserine at position 235 in the CH2 domain (IgG4-E), toremove residual binding to Fcg receptors, and substi-tution of serine with proline at position 228 in thehinge region (IgG4-PE) for greater stability. Pharma-cokinetic analysis in rats gave a t1/2 of approximately 4

ays for IgG4-E and 9 days for IgG4-PE, consistentith a greater stability of the IgG4-PE molecule. The

ffects on T cell subsets were assessed in chimpanzeesiven escalating doses of IgG4-PE: 0.05 mg/kg on Day6, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85.eceptor modulation was observed at the two highestoses, but no depletion of T cells at any dose. The initro and in vivo results demonstrate the potential ofhis IgG4-PE mAb for use in human trials. © 2000 Academic

ress

Key Words: CD4; monoclonal antibody; anti-CD4; Pri-matized; Fc region; chimpanzees; nondepleting; Tcells; clenoliximab.

INTRODUCTION

CD4 mAb therapy has been pursued in autoimmunediseases and transplantation to ameliorate the autode-structive role of CD41 T cells with the aim of sustain-ng a long-term clinical benefit. In this regard, CD4

Abs have shown promise in animal models and inlinical trials (1–7). During the elaboration of an im-une response, the CD4 molecule on the T cell surface

nteracts with MHC class II molecules on the antigen-resenting cell, resulting in signaling through its cyto-lasmic domain. Blockade of this interaction withoutisrupting the recognition of MHC peptide by the TCRan result in a T cell becoming tolerized or anergic to

1 To whom correspondence should be addressed.

1521-6616/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

164

antigen (3, 8–11). Contrary to initial thinking evidencein animal models shows that depletion is not neces-sary, and indeed may prevent sustained suppression ofspecific immune responses (11–14). Moreover, pro-longed and indiscriminate depopulation of CD4 T cellscarries the potential risk of increased susceptibility toinfectious agents.

Major inflammatory or autoimmune conditions thatcould benefit from the immunomodulation of the CD41

T cell compartment include rheumatoid arthritis, pso-riasis, and acute asthma. Approximately 1% of thepopulation suffers from rheumatoid arthritis, a diseasewith severe clinical and social consequences for themajority of these patients. The rheumatoid synoviumhas increased levels of CD41 T cells, which are hypoth-esized to both initiate and perpetuate the deleteriouseffects of inflammatory cells (15–18). Similarly the der-mal lesions of psoriasis are dominated by cell infil-trates of activated CD41 T cells (19, 20). Furthermore,injection of CD41 T cells in a SCID mouse engraftedwith symptomless psoriatic skin induced psoriaticplaques (21). T cell-suppressive agents such as cortico-steroids, cyclosporin A, CD3 mAbs, and CD4 mAbs areeffective in ameliorating symptoms, supporting the hy-pothesis that T cells play a central role in the patho-genesis of psoriasis.

We previously reported the development of CE9.1, orkeliximab, a Primatized IgG1 CD4 antibody (22, 23).This antibody was derived by immunization of cyno-molgus macaques with human CD4 and consists of theprimate V regions fused to human constant regions.Because of this derivation, keliximab only recognizesthe CD4 molecule of some higher primates (macaquemonkeys, baboons, and chimpanzees) and man. Due toits high level of sequence homology to human immu-noglobulin molecules, this antibody is expected to beimmunosilent in vivo (22, 23). Keliximab does show the

strong binding to Fcg receptors in vitro that is expected

lAardsa

165MODIFICATION OF ANTI-CD4 Fc REGION

for the IgG1 isotype. However, its binding to C1q issurprisingly weak and it is not active in CDC assays.This unusual property for an IgG1 antibody may ac-count for the only modest depletion of CD4 T cells inchimpanzees administered keliximab (23). Neverthe-less keliximab did induce substantial CD4 T cell deple-tion in human clinical trials even though clinical im-provement was seen in arthroscopic measurements ofpatients with resistant knee synovitis (24, 25). Wedescribe the construction of IgG4 derivatives of kelix-imab and their characterization in vitro and in vivowhich led to the selection of the PE variant (clenolix-imab) for development as a nondepleting CD4 mAb forhuman clinical trials.

MATERIALS AND METHODS

Molecular Construction and Expression of aPrimatized IgG4 Anti-CD4 Antibody

Human g4 heavy chain constant region was isolatedby RACE-PCR amplification from the cell line TPT10.4(S. Morrison, UCLA) using primers for the 59 and 39ends containing Nhe1 and BamH1 restriction sites,respectively. The PCR fragment was cloned into theexpression vector Anex 1 and sequenced. Anex 1 con-tained the variable region immunoglobulin (Ig) genesfrom the previously described anti-CD4 antibodyCE9.1, keliximab (23). The g4 heavy chain region fromTPT10.4 was cloned downstream of the existing VHregion and the sequence found to be identical to thatreported by Kabat et al. (26). The entire heavy andight chain immunoglobulin genes were subcloned fromnex 1 into the expression vector NEOSPLA 4 (22, 27)s a single BglII/SacI fragment. This construct waseferred to as the IgG4 derivative. For the IgG4-Eerivative, a glutamic acid was introduced at this po-ition by PCR using primers that incorporated a NheInd BspHI site at 59 and 39 ends, respectively. For the

IgG4-PE derivative, a PCR-amplified fragment con-taining two amino acid changes, a proline at position228 in the hinge and glutamic acid substitution atposition 235 in the CH2 region (EU numbering), wasintroduced into the IgG4 construct using primers thatincorporated NheI and BspHI sites at 59 and 39 ends,respectively. This IgG4-PE version is also referred toas clenoliximab (28, 29).

The three recombinant antibody constructs were ex-pressed in a similar manner by electroporation intoDG44 CHO cells followed by growth in selective media.The following description relates to the IgG4-PE con-struct. DG44 cells were grown in CHO-S-SFM II me-dium containing 50 mM hypoxanthine and 8 mM thy-midine, and 25 mg of NEOSPLA 3F plasmid, containingthe antibody construct, was electroporated into 4 3 106

DG44 cells using a BTX 600 electroporation device in

0.4-ml disposable cuvets. After electroporation, cellswere plated into 96-well dishes (40,000 cells/well) andfed with medium containing 400 mg/ml G418 (Geneti-cin, GIBCO) 2 days after electroporation and then ev-ery 2 or 3 days, conditions previously calibrated to giverise to single cell clones. After growth of G418-resistantcolonies (approximately 3–4 weeks), anti-CD4 anti-body was detected in the supernatants of 28 coloniesfrom five plates. One clone, 5C1, was selected andshown by Southern blot analysis to contain two copiesof the expression construct. To select for gene amplifi-cation, clone 5C1 cells were distributed into 96-wellplates, at concentrations from 1 3 106 to 3 3 104, andgrown in medium containing 5 nM methotrexate. Aftergrowth of cells in 5 nM methotrexate, supernatant wastested for the presence of anti-CD4 antibody in platesthat showed growth in less than 32 out of the 96 wells.This process was repeated at 50 nM, 250 nM, and 1 mMmethotrexate, ultimately yielding clone producing 35pg/cell/per day of CD4 mAb.

Fcg Receptor Binding Assay

The Fcg receptor binding assay measured the abilityof the antibody to bridge between two cell types, oneexpressing the CD4 molecule and the other expressingFcg receptors (23). Briefly, murine fibroblasts, trans-fected with human CD4 (DAP-CD4 cells), were allowedto adhere to flat-bottomed 96-well tissue culture platesovernight (2.5 3 105 cells/well/100 ml). FcgR bearingmonocytic THP1 cells (ATCC TIB202), which expressprimarily FcgRII but also low amounts of FcgRI andFcgRIII (unpublished data), were cultured for 18 h, ata density of 1 3 106 cells/ml, in media containing 50U/ml of IFN-g at 37°C and washed with Dulbecco’sPBS containing calcium, magnesium, and 0.1% bovineserum albumin. Cells were then incubated at 5 3106/ml with 20 mg/ml of the dye calcein acetomethoxyester (CAM, Molecular Probes, OR) according to themanufacturer’s instructions.

The various antibody constructs, at appropriate di-lutions, were incubated with DAP-CD4 cells in PBS for5 min. Dye loaded THP-1 cells (4 3 105 cells/well/50 ml)were then added to each well and incubated in thedark, at room temperature, for 1 h. Unbound cells wereremoved by gentle washing with PBS and the cellsremaining in the wells were lysed by the addition of 10ml 20% Triton X-100 (Sigma). Fluorescence was mea-sured at excitation/emission wavelengths of 485/538nm, using a Fluoroskan device (MTX Labsystems,McLean, VA) and Delta Soft software (BioMetallicsInc, Princeton, NJ).

Binding Affinity and Stoichiometry

Surface plasmon resonance. The binding of soluble

CD4 to immobilized antibodies was determined in sat-

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166 NEWMAN ET AL.

uration binding experiments by surface plasmon reso-nance (SPR) using a BIAcore apparatus (Pharmacia).The data for the association and dissociation phase ofthe SPR were analyzed directly using the rate equa-tions described (30) and the equilibrium binding con-stant was calculated.

Titration microcalorimetry. Titration microcalo-rimetry was used to determine the stoichiometry andaffinity of sCD4 binding to the various forms of theantibody. This method measures binding reactions byvirtue of their intrinsic heats of reaction, as describedpreviously (31). The method provides a high-precisiondetermination of the fraction of active sCD4 bindingsites on the mAbs. Since the sCD4 affinity of all mAbswas too high to measure directly by this method atambient temperature, a thermodynamic approach wastaken: (1) the affinity was measured at 45°C, where itis weak enough to be measured directly, and (2) thetemperature dependence of the binding enthalpy wasmeasured from 30 to 45°C. Together, these data al-lowed calculation of the affinity over a wide range oftemperature using the Gibbs-Helmholz equation (31)and the stoichiometry of sCD4 binding.

Antibody Stability

Differential scanning calorimetry (DSC). The tem-perature stabilities of IgG4-E, IgG4-PE, and IgG1 weremeasured by DSC (31), using a Microcal Inc., MC-2DSC. Unfolding temperatures were determined as thepeak of the unfolding transitions and compared foreach of the constructs to determine whether any of theantibodies differed in their stability to elevated tem-peratures.

Molecular stability of intact immunoglobulin bySDS-polyacrylamide gel electrophoresis (PAGE). Themolecular integrity of the intact IgG4 and its variantswas monitored by SDS-PAGE. Samples were run un-der nonreducing conditions to separate protein chainsthat were not covalently linked via disulfide bonds. Inaddition, a 3-month stability study on the relative per-centage of noncovalently linked heavy and light chainswas carried out after storage at 5 and 40°C. The per-centage content of noncovalently associated antibodydimer (“halfmer”) in each preparation was determinedby scanning Coomassie-stained gels after electro-phoretic separation.

Comparative PK Analysis of IgG4-E and IgG4-PE inthe Rat

Antibody was administered to male Spague-Dawleyrats at 1 mg/kg (4 animals per group) and blood sam-ples were taken for 4 weeks. Circulating plasma levels

of IgG4-E or IgG4-PE were determined using a sCD4/

anti-human Ig sandwich ELISA assay. In this assay100 ml of sCD4 (1 mg/ml) was coated onto a 96-wellmicrotiter plate, washed with water, and blocked with10% dried milk solution. Plasma was added (40 ml), atppropriate dilutions, for an incubation period of 60in. Any human Ig bound to sCD4 was then measured

y incubation with a horseradish peroxidase-labeledoat anti-human IgG antibody. This assay measuredot only the presence of circulating human IgG but also

ts ability to bind recombinant sCD4.

n Vivo Study in Chimpanzees

Based on the results obtained from the in vitro as-ays and the stability characteristics of the differentntibodies (Results), the IgG4-PE (clenoliximab) waselected for in vivo analysis in chimpanzees at the

Coulston Foundation (Alamogordo, NM). Six male an-imals were infused intravenously with increasingdoses of clenoliximab formulated in a buffer of 25 mMsodium citrate in saline, pH 6.5 containing 0.02%Tween 80. The dosing schedule is shown in Fig. 1.

Blood samples were taken from each animal, 3 and 2weeks prior to infusion of the antibody, to establishbaseline lymphocyte counts. On Day 1, each animalwas given an infusion of formulation buffer alone, asprevious experiments had shown that occasionally in-fusions of saline alone could affect CD41 cell counts.Each animal was given an antibody dose of 0.05 mg/kgon Day 16 followed by a 1.5 mg/kg dose on Day 43. OnDay 45 each animal was reinfused with saline and onDay 85 given the highest level dose of 15 mg/kg ofIgG4-PE. Antibody was diluted in formulation buffer,so that each dose was about 300 ml, given at a rate of150 ml per hour. Blood samples were taken at theintervals shown in Fig. 1. Parameters that were eval-uated included body weight, serum chemistry, hema-tology, urinalysis, and flow cytometric analysis of lym-phocyte subpopulations.

FIG. 1. Dosing schedule for intravenous infusion of clenoliximab.

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167MODIFICATION OF ANTI-CD4 Fc REGION

Flow Cytometry

The coating of CD41 T cells by antibody and the effecton CD41 T cell numbers were analyzed in whole bloodsamples by flow cytometry using standard procedures.All data were obtained using antibodies coupled to eitherfluorescein (FITC) or phycoerythrin (RPE) and examinedby double staining using a FACScan (Becton-Dickinson)flow cytometer. To stain lymphocytes for flow cytometry,5 3 105 white blood cells from whole blood were obtainedfter lysis of RBC using carbonate-buffered ammoniumhloride solution (0.17 M NH4Cl, 0.01 M KHCO3, 1 mM

tetrasodium EDTA, pH 7.3) and incubated on ice for 30min with appropriate antibodies as follows: anti-CD45-FITC to gate the lymphocyte population on a scatter plot;anti-CD4-FITC and anti-CD8-RPE to obtain CD4/CD8ratios; anti-CD3-FITC plus anti-CD4-RPE or anti-CD8-RPE for T cell counts; unconjugated IgG4-PE at a 10mg/ml saturating dose to determine maximal binding. Inaddition, the CD4 mAbs OKT4a and OKT4 were utilizedto assess the extent of CD4 antigen coating by clenolix-imab. OKT4A binds in domain 1 of CD4 and competeswith the binding of clenoliximab. Thus, the lack ofOKT4A binding indicates coating of CD4 on cells byclenoliximab. In contrast, OKT4 recognizes a noncompet-ing epitope in domain 4. Cells were washed once by cen-trifugation (200g, 6 min) with 2 ml cold PBS and incu-bated with antibody for 30 min on ice, and then they werewashed once, fixed in 0.5% paraformaldehyde, and storedat 4°C until analyzed. Flow cytometry data were acquiredon a Becton-Dickinson FACScan and analyzed usingWinList software from Verity Software House (ME). Theinstrument was set to auto-gating to allow examinationof quadrants containing cells that were single-stainedwith either RPE or FITC, unstained, or doubly stained.Lymphocytes were analyzed by which quadrant theyoccupied after flow cytometry and, in addition, themean fluorescence intensity (MFI) of the CD41 pop-ulation was examined to assess whether modulationor partial modulation of the CD4 molecule had oc-curred. Fluorescence intensity units were quanti-tated as molecules of equivalent soluble fluoro-chromes (MESF) using Quantum 26-FITCcalibration beads (Flow Cytometry Standards Corp.,San Juan, Puerto Rico). OKT4 and OKT4a were pur-chased from Ortho Diagnostics Systems, Inc., and allother conjugated antibodies were from Becton-Dick-inson. Total leukocyte populations were determinedusing a CellDyne 1600 automated hemocytometer.

Statistical Analysis

Statistical analysis of flow cytometry cell counts wasconducted on the following endpoints (measured as

cells/ml):

● Lymphocytes● Monocytes● Granulocytes● Total leucocytes● CD31 CD41

● CD31 CD82

● CD41 CD4A1

● CD31 CD42.

The design was a two-factor repeated-measures design;measures (endpoints) were repeated over dose and dayfor each experimental unit. The statistical analysisutilized a single-factor repeated-measures analysis ofvariance model (32) to construct a 95% confidence in-terval for the difference between treated and untreatedmean cell counts. Mean cell counts, which fell abovethe upper bound or below the lower bound of thisinterval, were considered statistically significant. Ex-ample ( y-axis 5 cell counts/ml): The confidence intervalwas constructed using the confidence interval for thedifference between means, as follows from the repeatedmeasures model.

ux# u 2 x# tu # ta,dfÎs e2S 1

nu1

1ntD ,

where x# u 5 mean of untreated observations (Days 228to 15, 64 to 85); x# t 5 mean cell count at any treatment

ay; nu 5 number of observations from which x# u wasetermined (nu 5 48); n t 5 number of observations

from which each x# t was determined (n t 5 6); se2 5 error

mean square from the analysis of variance statisticalmodel; and t 5 critical value of Student’s t distributionwith (m 2 1) 3 (d 2 1) degrees of freedom. In thequation, m 5 6 (number of monkeys), and d 5 18

(number of days in which an observation was re-corded). Since there are 11 days in which treated ob-servations were recorded (and thus 11 individual com-parisons to x# u), the overall probability of a falsepositive result was held to a 5 0.05 using Bonferroni’smethod (a 5 0.05/11; (33)); thus the value of Student’st in the equation above was set to t 5 3. The upper andlower bounds of the confidence interval were found byrearranging the equation above, as follows:

2ta,dfÎs e2S 1

nu1

1ntD # x# u 2 x# t # ta,dfÎs e

2S 1nu

11ntD[1]

x# u 2 ta,dfÎs e2S 1

nu1

1ntD # x# t # x# u 1 ta,dfÎs e

2S 1nu

11ntD .

[2]

CR

168 NEWMAN ET AL.

The lower confidence bound is given by the left-handside of Eq. [2]:

x# u 2 ta,dfÎs e2S 1

nu1

1ntD .

The upper confidence bound is given by the right-hand side of Eq. [2]:

x# u 1 ta,dfÎs e2S 1

nu1

1ntD .

When x# t falls above or below one of the bounds it isconsidered statistically significant, compared to x# u.

The above approach is a more sensitive test for de-pletion than the paired t test approach used by Lindseyet al. (34).

RESULTS

IgG4 Derivatives of Keliximab

To develop a keliximab derivative devoid of Fc effec-tor functions and therefore unlikely to cause CD4 T celldepletion, we first replaced the human g1 region withthat of g4 (IgG4 derivative). In comparison to g1, thehuman g4 isotype has a much reduced ability to fixcomplement and to bind to Fcg receptors (35). To in-vestigate a further reduction of the residual Fcg recep-tor recognition, leucine at position 235 in the CH2region of the g4 heavy chain (IgG4-E derivative) wasreplaced by glutamic acid. This substitution was based

TABLE 1

The Kinetics of CD4 Binding by Keliximab (IgG1) andlenoliximab (IgG4-PE), as Measured by Surface Plasmonesonance

Antibodyk a

(31026 M21 s21)k d

(3104 s21)KD

(nM)

IgG1 (keliximab) 1.1 6 0.2 2.1 6 0.5 0.19 6 0.05IgG4-PE (clenoliximab) 2.1 6 0.7 1.5 6 0.8 0.07 6 0.05

TABSummary of sCD4 Binding Thermodynamics of All Fo

AntibodyKD

a

(nanomolar)DG

(kcal/mol sCD4)

IgG1 (keliximab) 0.7 213.0IgG4 0.4 213.4IgG4-E 2.0 212.3IgG4-PE (clenoliximab) 1.3 212.6

a Uncertainty in KD is a factor of 2–3.

on the sequence of antibody subtypes with low Fcgreceptor binding activity. The consensus sequencethought to be primarily responsible for the binding ofcertain IgG subclasses to Fcg receptors is LLGGPS (35)and located at the N terminal end of the heavy chainCH2 region (EU numbering 234–239). The wild-typesequence for the IgG4 isotype contains a phenylalanineat position 234, giving the motif FLGGPS. The murineIgG2b isotype, which also has low Fcg receptor bindingactivity (35, 36), contains the sequence LEGGPS in thisregion. Thus, to potentially further reduce Fcg inter-action, we incorporated a glutamic acid residue into thehuman wild-type IgG4 sequence at position 235 to givethe sequence FEGGPS.

Due to the described instability of IgG4 antibodiesproduced in culture (37), we also replaced the serine atposition 228 in the hinge region of the IgG4-E deriva-tive with a proline (IgG4-PE). This modification intro-duces the CPPC motif present in the human IgG1hinge and was previously reported to stabilize the IgG4hinge (37). These IgG4 derivatives were produced inCHO cells and compared in their biophysical and func-tional properties, together with keliximab.

Binding Affinity and Stoichiometry

Table 1 shows the binding kinetics for CD4 bindingto keliximab and IgG4-PE, as measured by surfaceplasmon resonance (SPR). The on and off rate con-stants for the IgG1 and IgG4 constructs are not statis-tically different. Comparable results were obtainedwith the IgG4 and IgG4-E derivatives (unpublished).This demonstrates that changes in the Fc portion of themolecule did not affect antigen binding kinetics.

Measurements of the stoichiometry of sCD4 bindingto all four mAbs by titration microcalorimetry gave1.92 6 0.05 sCD4 per mAb, which indicated a highfunctional activity of the antibody preparations thathave a theoretical binding of two sCD4 molecules permolecule of mAb.

A summary of the sCD4 binding thermodynamics ofall four mAbs, as determined by titration microcalo-rimetry, is shown in Table 2.

2MAbs, as Determined by Titration Microcalorimetry

DHcal/mol sCD4)

2TDS(kcal/mol sCD4)

DC(kcal/sCD4/degree)

223 10 20.48221 8 N.D.221 9 20.42221 8 20.44

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169MODIFICATION OF ANTI-CD4 Fc REGION

The microcalorimetry results show that the affinitiesof all four mAbs were, within error, the same. More-over, the equivalent binding thermodynamics and ki-netics for all the mAbs argue that the molecular detailsof their sCD4 binding reactions are identical.

Thermal Stability

The temperature stability of the IgG4-E and -PEvariants was compared to that of the IgG1 parent bytemperature scanning microcalorimetry. The unfold-ing of all the antibodies occurred in a single transitionwith a Tm of 70°C. The unfolding was irreversible sug-gesting that the structural forces maintaining the mo-lecular integrity of the molecule are similar for each ofthe constructs.

Molecular Stability

The physical and chemical stability of the IgG4-Eand IgG4-PE constructs were monitored over 3 monthsat 5 and 40°C, and diffused light by SDS-PAGE (re-duced and nonreduced), IEF, reverse phase-HPLC,size-exclusion chromatography (SEC), and ELISA. Inagreement with the thermal stability analysis, bothantibodies showed similar and minimal degradation bySEC, IEF, HPLC, and ELISA (not shown). However,consistent with a prior report (38), SDS-PAGE re-vealed a striking difference between these antibodies.Under reducing conditions, the expected heavy andlight chain fragments were observed for both antibod-ies (not shown). However, under nonreducing condi-tions, the IgG4-E construct showed a prominent 75- to80-kDa species in addition to the expected 150-kD spe-cies corresponding to the full covalently associatedheavy and light chain dimer. This halfmer, correspond-ing to a single heavy and light chain, molecule was alsopresent in the wild-type IgG4 derivative (not shown)and demonstrates that the hinge disulfide bonds form

FIG. 2. SDS-PAGE analysis of IgG4-E and IgG4-PE run undernonreducing conditions. Lane 1, IgG4-PE batch 1. Lanes 2–4, IgG4-Ebatches 1, 3, and 4, respectively. Lane 5, IgG4-E stored for 3 monthsat 5°C. Lane 6, IgG4-E stored for 3 months at 40°C. Lane 7, IgG4-PEstored for 3 months at 5°C. Lane 8, IgG4-PE stored for 3 months at40°C.

inefficiently in these recombinant antibodies. This de-

fect is corrected by the proline substitution at position228, giving the middle hinge sequence CPPC that ispresent in IgG1 heavy chains.

The halfmer content of IgG4-E and IgG4-PE wasmonitored in samples purified from different lots todetermine the batch-to batch variability. In four differ-ent preparations of the IgG4-E variant, the fraction ofhalfmer ranged from 9 to 28%, whereas it was less than1% in the two preparations of IgG4-PE. The extent ofhalfmer in a specific preparation of IgG4-PE antibodydid not change substantially over a period of 3 monthsat either 5 or 40°C (Fig. 2), indicating that is was astable property of the preparation. A specific IgG4-Epreparation showed some increase with time at both 4and 50°C. The absence of the hinge disulfide bonds in aportion the IgG4-E sample did not impact the overallkinetic and thermodynamic properties of this antibody(above).

Fcg Receptor Binding Properties of EngineeredAntibodies

To approximate in vivo conditions, we utilized a Fcgreceptor binding assay that measured the ability of theCD4 antibody to form a bridge between CD4 and Fcgreceptor-expressing cells. Figure 3 shows that, surpris-ingly, the wild type IgG4 derivative showed identicalbinding characteristics to the IgG1 parent (keliximab).The curve showed typical binding kinetics with a pla-teauing of binding between 100 and 1000 ng/ml. Bycontrast, the IgG4E version, containing the glutamicacid 235 change in the CH2 region, showed no binding,even at the maximum concentration used, 1 mg/ml.

The lack of Fc receptor binding by IgG4-E andIgG-PE derivatives was confirmed in other assays re-ported elsewhere (38). We conclude that the IgG4 iso-type, although generally considered a weak Fcg recep-

FIG. 3. Binding of IgG1, IgG4, and IgG4E versions of PrimatizedCD4 antibody to Fcg receptors on THP1 cells in an FcgR-CD4-

mediated cell-cell adhesion assay.

170 NEWMAN ET AL.

tor binder, is capable of interacting with Fcg receptorsunder certain conditions. The substitution of leucine235 by glutamic acid, however, eliminates this residualFcgR binding.

Pharmacokinetic Studies in Rats

Both the IgG4-E and IgG4-PE derivatives of kelix-imab possessed the desired in vitro characteristics for atherapeutic antibody, i.e., a lack of effector functionand the ability to bind, with high affinity, to CD4.However, since the IgG4-E derivative showed high andvariable levels of halfmer content, both antibodies wereexamined for their pharmacokinetic properties in rats.

Following a 1 mg/kg intravenous bolus administra-tion of CD4 mAb, the plasma concentrations of IgG4-Edeclined in a triphasic manner, and the plasma con-centrations of IgG4-PE declined in a biphasic manner(Fig. 4).

For comparative purposes, all plasma concentration-time profiles were analyzed using a biphasic model.The predominant secondary phase t 1/ 2 was approxi-mately 4 days for IgG4-E, and 9 days for IgG4-PE, andaccounted for 67 and 93%, respectively, of the totalarea under the plasma concentration-time curve. Oneanimal (No. 4) showed an immune response to thehuman IgG4-PE antibody and the plasma concentra-tions of circulating IgG4-PE declined rapidly after300 h. The apparent plasma clearance for IgG4-PE waslow (0.4 ml/h/kg) and was approximately half the clear-ance of IgG4-E (1.0 ml/h/kg). The long circulating half-life of functionally intact IgG4-PE in the rat suggeststhat it is likely to be effective over an extended periodof time when administered to man.

Effect of IgG4-PE (Clenoliximab) on ChimpanzeeCD41 T Cells in Vivo

Although the IgG4-E and IgG4-PE antibodies exhib-ited similar properties in in vitro functional tests, CD4binding activity, and stoichiometry, the IgG4-PE ver-sion showed greater molecular integrity of interchaindisulfide bond formation. However, based on pharma-cokinetic properties observed in the rat, the serumhalf-life of IgG4-PE is more than twice that of theIgG4-E. Based on these characteristics, IgG4-PE(clenoliximab) was selected to determine the effect ofadministration on CD41 and other T cell subsets inchimpanzees. Blood samples were taken, as indicatedin Fig. 1, analyzed by flow cytometry, and used tocompile data of CD41 T cell numbers and IgG4-PEcoated CD41 T cells. Figure 5a shows the numbers ofcirculating CD31/CD41 cells after increasing doses ofIgG4-PE. At the lower doses, the CD41 cell population

remained within the normal range, as shown in the

figure by the upper and lower 95% confidence limits. Atthe highest dose (15 mg/kg) there was an apparentdecrease in circulating CD4 cells. However, when thenumber of CD4 cells was measured indirectly as CD31/CD82 cells, CD4 cell numbers did not decrease at anydose (Fig. 5b). Indeed, there was a slight increase inthe number of CD31/CD82 cells after the 15 mg/kg dosethat persisted for 6 days (Days 87–92) and which isunexplained (Fig. 5b). A corresponding rise in lympho-cyte count (CD451) was observed after the 15 mg/kgdose, Fig. 5f, and persisted for 20 days (Days 87–106).However, the total leukocyte count was unaffected(data not shown).

The CD81 lymphocyte population was not affected byadministration of CD4 antibody at the lower doses, butafter the 15 mg/kg dose, there was a rise in CD81 cellcounts (Fig. 5c), similar to that seen in the CD31/CD42

population (an indirect way of measuring the CD81

population). The lymphocyte hyperplasia after thehighest dose of antibody, therefore, was reflected inelevated levels of both CD41 and CD81 lymphocytesubsets. Other cell types, such as monocytes and gran-ulocytes, did not change (data not shown) and sincethey represent the overwhelming majority of the cellsin the differential count, the elevation in lymphocytecount was not reflected as an elevation in the totalleukocyte count.

The coating of CD4 cells with the IgG4-PE antibodywas assessed with CD4 mAbs OKT4 and OKT4A.OKT4A competes with IgG4-PE whereas OKT4 bindsto a distal noncompeting site on the CD4 molecule.Therefore, plotting the number of cells that were bothOKT41 and OKT4A1 gives an indirect measure of thenumber of cells coated with IgG4-PE, Fig. 5d. Two daysafter infusion, 90 and 100% of the CD41 cells were

FIG. 4. Change in serum concentrations of IgG4-E and IgG4-PEantibody with time following a 1 mg/kg bolus injection of antibodyinto rats.

coated with antibody at the 1.5 and 15 mg/kg doses,

l ly

171MODIFICATION OF ANTI-CD4 Fc REGION

respectively, with little coating after the 0.05 mg/kgdose. The percentage of antibody coated CD41 T cellsreturned to baseline after 12 days at the 1.5 mg/kgdose, whereas at least 50% antibody coating persistedfor 20 days at the 15 mg/kg dose.

In addition to recording the number and percentagesof CD41 and CD31/CD82 cells, the mean intensity of

FIG. 5. Circulating blood leucocyte counts after administrationValues represent means of values taken from six individual animalsCells lying within these boundaries exhibit normal fluctuation. Medensity. (a) Circulating CD31OKT41 cells; (b) circulating CD31CD82

cells; (e) mean intensity of fluorescence MFI of OKT41 cells; (f) tota

fluorescence of the CD41 fraction was also examined.

The MFI of cells in the CD31/CD41 quadrant, whenplotted against dose level, showed a reduction in MFI,directly after administration of the 1.5 and 15 mg/kgdoses but not after the 0.05 mg/kg dose, Fig. 5e. Asthere was no change in the number of CD31/CD82 cellsthroughout the antibody treatment, Fig. 5b, we inter-preted this decrease in CD4 MFI as a modulation of cell

ncreasing doses of IgG4-PE to chimpanzees at Days 16, 43, and 85.rizontal lines represent the upper and lower 95% confidence limits.

fluorescence intensity (MFI) is used as a measure of CD4 receptorlls; (c) circulating CD31 CD81 cells; (d) circulating OKT41 OKT4A1

mphocyte count.

of i. Hoan

ce

surface CD4 molecules.

dacduw

G

mctch

172 NEWMAN ET AL.

In summary, this study in chimpanzees showed thatsaturation of the CD4 molecule on the surface of circu-lating CD41 T lymphocytes was nearly complete at a

ose of 1.5 mg/kg and was complete and longer lastingt a dose of 15 mg/kg. Although there was no signifi-ant depletion of the CD41 T cell population at anyose, a substantial fraction of CD4 molecules was mod-lated from the cell surface after high density coatingith the IgG4-PE antibody.

ross Observation and Clinical Pathology

None of the animals in the study showed any abnor-alities in gross analysis including body weight, food

onsumption, behavior, and dermatological, gastroin-estinal, cardiovascular, or ocular examination. Serumhemistry values all fell within normal ranges, as didematological findings and urinalysis.

DISCUSSION

Keliximab is a Primatized CD4 mAb of the humanIgG1 isotype that consequently recognizes CD4 in onlyman and higher primates. This antibody is a potentinhibitor of CD4-mediated cell function in vitro andshows surprisingly weak interaction with complementreceptors (22, 23). However, in clinical studies, thisantibody did induce CD4 cell depletion (28, 29), anactivity presumed to be mediated through its high af-finity binding to Fcg receptors. Toward the goal ofreducing this cell-depleting activity, the present studydescribes the generation and characterization of threeIgG4 derivatives of keliximab. The IgG4-PE derivative,containing proline and glutamic acid substitutions atresidues 228 and 235, respectively, was not only devoidof Fcg receptor binding activity, but was stable in vitroand in vivo, having a 9-day half-life in the rat. In thechimpanzee, this antibody showed CD4 receptor mod-ulation but did not result in depletion of CD41 T cells.In other studies (38) this antibody displayed the samein vitro functional potency as keliximab in inhibition ofMLR. Thus, this IgG4-PE derivative, now namedclenoliximab, has the desired profile for clinical studywith a nondepleting CD4 mAb.

Components of the complement system possess theability to interact with the constant regions of certainantibody classes in a manner that leads to cell lysis anddestruction. The IgG1 isotype in humans classicallybinds complement components well, whereas thegamma 4 isotype lacks the ability to bind C1q. How-ever, the ability to bind complement is not necessaryfor cell depletion in vivo (39). In the present case, boththe IgG1 and the IgG4 versions of this CD4 mAb do notfix complement or bind C1q. Human CD41 T cells,

therefore, could become targets of cytotoxic FcgR bear-

ing effector cells in vivo, once they have been coatedwith an anti-CD4 antibody. Although the wild-typeIgG4 isotype binds Fcg receptors weakly, compared tothe IgG1 isotype, we show that it is not totally devoidof Fc binding activity. Others have shown that anunmodified IgG4 MAb can cause cell depletion in vivo(40). The sequence reported to be primarily responsiblefor the binding to Fcg receptors has been defined asLLGGPS (35). This sequence, located at the N terminalend (EU numbering 234–239) of the heavy chain CH2region, is conserved in human IgG1, IgG3, and murineIgG2a isotypes, all of which bind Fcg receptorsstrongly. The wild-type sequence for the IgG4 isotypecontains a phenylalanine at position 234, giving themotif FLGGPS. The murine IgG2b isotype, also a poorbinder of Fcg receptors, contains the sequenceLEGGPS. In constructing IgG4-PE (clenoliximab),therefore, we incorporated the glutamic acid residueassociated with murine IgG2b into the human wild-type IgG4 CH2 domain to give the sequence FEGGPS.This modification was successful in reducing CDC (38)and ADCC activities and virtually eliminated bindingto FcgRI and FcgRII in vitro.

In addition to the introduction of glutamic acid, thereplacement of serine 228 by a proline, resulted in amolecule that was more stable than the wild-typeIgG4. The IgG4 molecule tends to show inefficient for-mation of interchain disulfide bonds in the hinge re-gion. The introduction of a proline, as originally de-scribed by Angal et al. (37), is thought to providerigidity to the hinge and promote more efficient inter-chain bonding. Possibly, the presence of a serine atposition 228 promotes preferential linkage of intra-chain rather than inter-chain disulfide bonds by neigh-boring cysteine molecules. The resulting CPPC motif inthe middle hinge region of the IgG4-PE derivative isthe same as that of the IgG1 parent, keliximab.

Although the in vitro data showed no apparent CD41

receptor modulation, the in vivo studies revealed apartial CD4 modulation after 1.5 mg/kg dose and vir-tually complete receptor modulation after 15 mg/kgdose of clenoliximab (Fig. 5). When the CD31CD41

fraction was analyzed, the intermediate dose of 1.5mg/kg showed no drop in the number of CD31CD41

cells; this was only seen at the 15 mg/kg dose. Both the1.5 and the 15 mg/kg doses did show a reduction inMFI. This can be interpreted as partial modulation ofCD4 molecules at the 1.5 mg/kg dose but not sufficientenough that the CD31CD41 cell fraction fell outsidethe CD31CD41 quadrant by flow cytometry. At thehighest dose of 15 mg/kg, there was a combination ofcells that fell outside the CD31CD41 quadrant andcells that remained inside this quadrant but had amuch reduced MFI. Thus, some cells had undergonepartial modulation and others complete modulation,

although the cell numbers themselves were un-

2

2

2

173MODIFICATION OF ANTI-CD4 Fc REGION

changed. Despite the cell surface modulation of theCD4 molecule, no depletion of T cells was detected inany of the animals, since the percentage of CD31/CD82

T cells was unchanged from baseline CD41 T cell pop-ulations for each animal.

The mechanism of receptor modulation is unknown.Removal of Fcg receptor binding activity and failure toobserve modulation in vitro, even in the presence ofFcg receptor bearing monocytes, suggested that Fcgreceptor binding to monocytes may not be the onlymechanism in vivo. Other evidence from preclinicaland clinical studies with CD4 mAbs suggests thatstripping of the CD4 molecule can account for part ofthe down modulation which has been observed for CD4mAbs (28).

In summary, simple molecular exchange of an IgG1constant region for an IgG4 alone did not result incomplete incorporation of all the desired characteris-tics for an efficient therapeutic anti-CD4 antibody. Mo-lecular integrity of the disulfide bridges required asingle amino acid substitution in the hinge region andelimination of residual Fcg receptor binding of theIgG4 heavy chain required a second single amino acidsubstitution in the CH2 region. The final constructIgG4-PE was shown to be nondepleting but did modu-late CD4 from the cell surface. This antibody has sub-sequently been given the USAN name “clenoliximab”and entered human trials for the treatment of rheuma-toid arthritis.

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Received July 19, 2000; accepted with revision October 17, 2000; published online December 6, 2000