Rheumatology 2003; 42: 54-61
© 2003 British Society for Rheumatology
Antibody-mediated stripping of CD4 from lymphocyte cell surface in patients with rheumatoid arthritis
Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, King of Prussia, PA and
1 IDEC Pharmaceuticals, San Diego, CA, USA
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Abstract |
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Objective. Keliximab studies have provided evidence of the therapeutic potential of a non-depleting CD4 monoclonal antibody (mAb) in the treatment of rheumatoid arthritis (RA). Clenoliximab, an immunoglobulin G4 derivative of keliximab, has substantially reduced potential to deplete CD4 cells. In initial studies of clenoliximab, we investigated the hypothesis that the decrease in cell surface CD4 is the result of antibody-mediated stripping from the cell surface.
Methods. Patients received single or multiple intravenous infusions of clenoliximab as follows: 0.05, 0.2, 1, 5, 10 or 15 mg/kg (n=3–5/group); 150 or 350 mg weeklyx4; or 350 or 700 mg every other weekx2 (n=12/group). Blood was collected for up to 16 weeks and pharmacokinetic and pharmacodynamic assessments were conducted using immunoassay and flow cytometry.
Results. CD4 count was largely unaffected by clenoliximab treatment. Dose-dependent CD4 coating, down-modulation and stripping were observed. Maximal down-modulation persisted for an increasing period as dose increased, while soluble CD4–clenoliximab complexes accumulated. The amount of CD4 in soluble complex was as much as 20 times the amount of cell-associated CD4. For the same total dose, administration of higher doses, less frequently, resulted in pharmacodynamic profiles similar to those of lower doses administered more frequently.
Conclusion. Decrease in the density of CD4 on the T-lymphocyte surface is caused by antibody-mediated stripping.
KEY WORDS: CD4, Monoclonal antibodies, Pharmacokinetics, Pharmacodynamics, Receptor stripping, Rheumatoid arthritis, T cell.
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Introduction |
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CD4 is a membrane-bound glycoprotein antigen expressed predominantly on lymphocytes, including peripheral helper/inducer T cells. CD4 is critical for interaction with antigen-presenting cells bearing class II major histocompatibility complex (MHC II) molecules. This interaction results in enhancement of the immune response [1]. Antibodies to CD4 have been shown to inhibit T-cell proliferation and cytokine production in vitro [2, 3] and have been studied in patients with rheumatoid arthritis (RA), in which T-cell activation plays a crucial role in inflammation and tissue damage (e.g. 4). CD4 monoclonal antibodies (mAbs) mediate their in vivo immunomodulatory effects via mechanisms that may include (i) depletion of CD4+ T cells from the systemic circulation, peripheral lymphoid organs and/or sites of inflammation, (ii) down-modulation of CD4 (decrease in cell surface density), and/or (iii) inhibition of CD4–MHC II interactions.
Clenoliximab (IDEC-151, SB-217969) is a chimeric macaque/human CD4 mAb of immunoglobulin (Ig) G4 isotype that inhibits antigen-induced T-cell proliferation, lymphokine release and helper T-cell functions [5, 6]. Clenoliximab has the same antigen-combining site as keliximab, an IgG1 mAb that showed promise in the treatment of RA in phase II/III clinical trials [7]. However, clenoliximab has greatly reduced Fc receptor affinity as a result of the IgG4 isotype and several key amino acid substitutions in the constant heavy-chain domain 2. Clenoliximab therefore has reduced potential to deplete CD4+ T cells in vivo while inhibiting T-cell activation through antigen coating and down-modulation.
In single-dose studies in patients with active RA, clenoliximab caused dose- and time-dependent CD4 coating and down-modulation with no significant depletion of CD4+ T cells [8]. Herein we describe the pharmacokinetics and pharmacodynamics from the first multiple dose study of clenoliximab in patients. Analysis of soluble CD4 (sCD4)–clenoliximab complexes in serum from patients in both the single- and multiple-dose studies was performed. The data suggest that down-modulation is caused by antibody-mediated stripping of CD4 from the lymphocyte cell surface. An understanding of the temporal relationships between drug concentration, coating, stripping and down-modulation has provided insight regarding both the optimization of dosing schedules for longer-term treatment and the in vivo mechanism of action of clenoliximab.
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Methods |
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Study designs
The single-dose study was a double-blind, placebo-controlled, randomized, single ascending-dose, parallel study performed on 32 adult patients with moderate to severe RA [8]. The patients were predominantly female (75%) and Caucasian (92%). The majority of patients tested positive for serum rheumatoid factor and were taking a stable dose of steroid. The median baseline absolute CD4+ T-cell count was 969 cells/µl. The mean patient age was 51 yr and mean weight 82 kg.
Six dose groups were studied: 0.05, 0.2, 1.0, 5.0, 10.0 and 15.0 mg/kg. There were three to five subjects in each active dose group and eight patients received placebo. Clenoliximab was administered in the morning as a single 2-h intravenous (i.v.) infusion into the cubital vein. Blood samples were obtained by direct venipuncture in the arm contralateral to the infusion site. Blood samples (
10 ml) for the determination of mAb and complex concentration were obtained immediately prior to drug administration (0 h) and 1, 2 (end of infusion), 3, 4, 24, 48, 72 and 96 h after the start of the infusion. Additional blood samples were collected approximately 1, 3, 4, 5, 7 and 12 weeks after dosing. Blood samples (3 ml) for flow cytometry were obtained 0, 2, 24, 48, 72 and 96 h and 2, 4, 6, 8 and 12 weeks after the start of dosing.
A similar patient population was studied in the repeat-dose protocol. This was a multicentre, double-blind, placebo-controlled, randomized study performed on 60 patients. Multiple i.v. infusions of placebo weekly, 150 mg weekly, 350 mg weekly (total of four doses), 350 mg every other week or 700 mg every other week (total of two doses) were administered over 2 h (n=12 per group). The groups dosed every other week received placebo in alternate weeks. Drug administration and blood sampling were performed as in the single-dose study except that blood samples were collected as follows: prior to dosing and 2 h after dose (end of infusion) for the weekly and alternate-week regimens, then weekly during follow-up for a total of 16 weeks.
Flow cytometry
Peripheral blood lymphocyte populations were analysed using an Ortho Cytoron Absolute (Ortho Diagnostic Systems Inc., Raritan, NJ, USA) flow cytometer. In this assay, cell surface membrane-bound proteins in whole blood were stained with a range of different fluorescence-labelled antibody probes. After a 20-min incubation period, the red blood cells were lysed, leaving the antibody-coated white cells intact.
Immunophenotyping analysis included determination of the absolute number of CD4+ cells (using the OKT4 mAb) and the mean fluorescence intensity (MFI) from cells identified as CD4+. The latter value was proportional to the receptor density on a CD4+ cell. Postdose MFI values were expressed as a percentage of the predose value. The absolute values of the MFI in predose samples were very similar [mean 25.3 (S.D. 1.5) arbitrary units in the single-dose study].
The OKT4 reagent used in these analyses binds to an epitope on the CD4 molecule that is distinct from the epitope recognized by clenoliximab. These measurements, therefore, were not expected to be affected by the presence of clenoliximab. The percentage T-cell coating by clenoliximab was assessed by comparison of the absolute CD4+ T-cell count obtained using OKT4 and the cell count obtained using Leu3a mAb. Leu3a binds to the same epitope as clenoliximab and cannot detect T cells in the presence of clenoliximab.
Immunoassays for clenoliximab and sCD4–clenoliximab complex
Serum concentrations of clenoliximab were determined using an electrochemiluminescent immunoassay (ECLIA) based on the binding of clenoliximab to recombinant sCD4 [8]. The assay response was generated by complexation of the analyte with biotinylated recombinant sCD4 (produced in-house), streptavidin-conjugated paramagnetic beads and a ruthenium-labelled mouse anti-human mAb (CALTAG Laboratories, Burlingame, CA, USA) specific for the CH3 domain of human IgG4 [9]. The lower limit of quantification was 100 ng/ml in neat human serum.
Soluble CD4–clenoliximab complex concentration was estimated using an enzyme-linked immunosorbent assay (ELISA) or an ECLIA. The ECLIA had a broader dynamic range and greater throughput. Both assays employed reagents with identical specificity. In the ELISA format, the complex was captured using the OKT4 mAb (with specificity for the sCD4 portion of the complex) and detected using the mouse anti-human IgG4 mAb (with specificity for the clenoliximab portion of the complex). In the ECLIA format, the assay response was generated by complexation of the analyte with biotinylated OKT4, streptavidin-conjugated paramagnetic beads and the ruthenium-labelled mouse anti-human IgG4 mAb.
In both assay formats, sCD4–clenoliximab complex concentration values were interpolated from a calibration curve prepared by mixing recombinant sCD4 and clenoliximab in a 2:1 molar ratio. The lower limit of quantitation was 6 ng/ml in neat human serum (expressed in terms of the amount of CD4 in the standard complex).
Western blot analysis
OKT4 was immobilized on an AminoLinkTM agarose column from Pierce (Rockford, IL, USA) by reductive amidation with sodium cyanoborohydride at pH 10, according to the manufacturer's directions. The affinity column was washed with 50 mM sodium phosphate, 0.75 M sodium chloride, pH 7.4, then equilibrated in 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (phosphate-buffered saline). Two millilitres of serum from each of five patients participating in the single-dose study were pooled then mixed with an equal volume of phosphate-buffered saline. These samples contained 100–300 ng/ml sCD4–clenoliximab complex by immunoassay. After the sample had been loaded on the column, the column was washed extensively with 50 mM sodium phosphate, 0.75 M sodium chloride, pH 7.4, and then with phosphate-buffered saline. sCD4 was eluted in 2 M ammonium hydroxide then lyophilized.
Purified patient sCD4 was solubilized in sodium dodecyl–sulphate polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer. SDS–PAGE was performed with a 4–20% gradient gel. Analyte was detected using a rabbit polyclonal antibody specific for human CD4 (produced in-house) and horseradish peroxidase-conjugated donkey anti-rabbit polyclonal antibody. 3,3',5,5'-tetramethylbenzidine chromogenic reagent (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was used for visualization according to the manufacturer's directions.
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Results |
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Single-dose administration of clenoliximab
Concentration–time profiles of serum clenoliximab concentration, CD4 coating, CD4 modulation and sCD4–clenoliximab complex for two patients in the 1 and 10 mg/kg groups are depicted in Fig. 1
for comparison. Serum concentration–time profiles were convex (Fig. 1A). There was an initial period during which concentrations decreased relatively slowly, and this was followed by a period of increased mAb clearance. This was less pronounced at 1 than at 10 mg/kg. Coating of CD4 on the cell surface was complete (100%) at the first time-point after drug administration and for all subsequent time points at which free mAb concentration exceeded 100 ng/ml (Fig. 1B). When drug concentrations were <100 ng/ml (below the assay limit of detection), CD4 coating was not detected. Thus, the duration of complete antigen coating was the same as the time period over which the free mAb concentration was quantifiable (3 days at 1 mg/kg and 20 days at 10 mg/kg) for these two patients.
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) clenoliximab to two patients. Maximal CD4 coating (100%) and maximal down-modulation (to 30% of baseline) were maintained, while sCD4–clenoliximab complex accumulated, until clenoliximab was cleared from the circulation.Immediately following dosing, when mAb serum concentrations were highest and CD4 antigen coating was complete, only small decreases in CD4 density were observed initially (Fig. 1C
) and sCD4–clenoliximab complex was undetectable (Fig. 1D). sCD4–clenoliximab complex was similarly not detected in any patient in the 0.05 (n=3) and 0.2 mg/kg (n=3) dose groups. For the patient depicted who received 1 mg/kg, both maximal down-modulation to 30% of baseline and maximal complex concentrations of 40 ng/ml were observed 2 days after dosing. (Maximal complex concentration was 20 ng/ml in the other patient in the 1 mg/kg dose group; in the remaining two patients insufficient sample was available for analysis). MFI returned to predose levels 4 days after dosing; at this time-point, complexes were no longer detectable.
In the patient depicted who received 10 mg/kg clenoliximab, maximal down-modulation, again to 30% of baseline, was observed 24 h after dosing and the decreased MFI persisted for several weeks. sCD4–clenoliximab complex concentrations increased throughout this time period to a maximum of 130 ng/ml (Fig. 1D). sCD4–clenoliximab complex was present in all the patients in the 5, 10 and 15 mg/kg dose groups (total of 12 patients; in the remaining two patients insufficient sample was available for analysis). Maximal complex concentrations ranged from 30 to 130 ng/ml in the 5–15 mg/kg dose groups. MFI returned to baseline in a time frame similar to, but consistently longer than, the duration of saturable coating (5 weeks at 10 mg/kg); complex was measurable only in the presence of free mAb (Fig. 1C and D). Generally, over time, maximal CD4 coating (100%) and maximal down-modulation (to 30% of baseline) were maintained, while sCD4–clenoliximab complex accumulated. Clearance of clenoliximab from the circulation was accompanied by clearance of the complex and delayed recovery of cell-surface CD4 density.
Despite the high initial serum concentrations following mAb infusion, complex was undetectable in all patients up to 4 h after initiation of the 2-h infusion. This indicated there was no pre-existing sCD4 in these patients and that accumulation of the complex required some time. Due to limited blood sampling, the decline in complex concentration was not fully characterized in most patients. However, as noted above, when clenoliximab decreased to non-quantifiable concentrations, sCD4–clenoliximab complex was no longer detectable in serum. Western blot analysis (Fig. 2) demonstrated that the sCD4 component of the circulating complex was approximately the same size (45 000 Da) as recombinant sCD4. Recombinant sCD4 has the transmembrane and intracellular C-terminal domains deleted relative to the full-length membrane protein. No species of lower molecular weight were detected.
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Multiple-dose administration of clenoliximab
As was observed following a single dose [8], multiple i.v. infusions of clenoliximab did not affect the number of circulating CD4+ T cells in patients (Fig. 3).
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For patients receiving weekly infusions of 150 mg clenoliximab, drug was non-quantifiable within 1 week of each dose administration. sCD4–clenoliximab complexes were largely non-quantifiable (n=6; in the other six, insufficient sample was available for analysis) and moderate CD4 down-modulation was observed for this group (n=12). It is likely that maximal down-modulation and stripping did occur for this dose level but was not captured with the infrequent, weekly blood-sampling scheme employed. With multiple administrations of higher doses of clenoliximab, the amount of CD4 stripping was variable. sCD4–clenoliximab complex was largely undetected in five of 12 patients in the 350-mg group weekly and in four of 12 patients in the 700-mg alternate-week groups, despite significant drug exposure and maximal CD4 down-modulation.
In the remaining patients in the higher dose groups (350 mg weekly, 350 mg every other week and 700 mg every other week), sCD4–clenoliximab complex detection was linked temporally with CD4 down-modulation, as in the single-dose study. As in the single-dose study, up to 2 h after the first dose the complex was not detected and CD4 down-modulation was minimal. Concentration–time profiles of the serum clenoliximab concentration, CD4 modulation and sCD4–clenoliximab complex for individual patients from selected multiple-dose regimens are depicted in Fig. 4 for comparison.
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) and sCD4–clenoliximab complex (
) concentration–time profiles following multiple i.v. infusions of 350 mg weekly (A), 350 mg every other week (B) or 700 mg every other week (C) to three patients. Slight accumulation of free mAb was observed for the 350 mg weekly (A) and 700 mg alternate-week (C) regimens. Down-modulation was maximal within 1 week for all regimens; however, between doses of the 350-mg alternate-week regimen (B), clearance of drug (detection limit represented by dashed line), recovery of CD4 density and clearance of complex was evident. Antigen density remained maximally depressed throughout treatment for the regimens shown in A and C, with no additive effect of repeat dosing on residual CD4 density.Weekly infusions of 350 mg clenoliximab resulted in moderate accumulation of free mAb, with concentrations exceeding 10 µg/ml throughout the month of treatment for the individual depicted in Fig. 4A
. In only two of 12 patients was clenoliximab cleared between any two doses for this regimen. CD4 density was maximally depressed 1 week after dosing and for the following month of treatment in this individual. Additional doses did not result in further decrease in CD4 cell-surface density but delayed recovery relative to single administration. sCD4–clenoliximab complex was detected at 1 wk then throughout treatment for this individual (Fig. 4A). Six of the seven patients with detectable complex in this group behaved similarly; for the other patient, neither drug nor complex was detected at selected troughs. Complex concentration appeared to increase modestly over time throughout the treatment period and was cleared by the time free mAb was cleared.
Following administration of 350 mg clenoliximab every other week, for the individual depicted, free mAb was cleared between doses and CD4 cell-surface density recovered partially (Fig. 4B). Complex was cleared between doses for this regimen as well (n=10; for the other two patients insufficient sample was available for analysis or the patient inadvertently received a different regimen). Interestingly, there was much more complex generated by the 1-wk time-point for this patient than by 1 wk for the patient administered 350 mg weekly. A dose of 700 mg every other week was enough to prevent mAb clearance between doses in eight of 11 patients and to decrease CD4 density persistently in 10 of 11 patients (Fig. 4C). The CD4 MFI–time profile was very similar for 350 mg weekly and 700 mg every other week. Also, the complex concentration–time profile was very similar for the patients depicted who received 350 mg weekly or 700 mg every other week (Fig. 4A and C).
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Discussion |
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We propose that down-modulation is caused by antibody-mediated stripping of CD4 from the T cell surface. There are many examples of receptors that shed an extracellular domain in response to ligand binding [10, 11]. There is also precedence for mAb treatment resulting in the detection of soluble antibody–antigen complexes when the antigen exists in both cell-associated and soluble forms [12]. However, we have found no example of soluble antibody–antigen complex formation, following mAb treatment, in which the cell-associated antigen does not normally exist in soluble form, as in the present case.
As CD4 is a single polypeptide chain with one transmembrane domain and clenoliximab does not deplete CD4 cells in patients, we hypothesize that stripping is the result of peptide bond hydrolysis. This may be enzymatically mediated, or the possibility exists that antibody complexes on the cell surface may be susceptible to non-enzymatic hydrolysis. sCD4 was observed previously in patients receiving a murine mAb to CD4. However, in this case, the antibody depleted CD4+ T cells, so the sCD4 detected may have been the result of cell killing [13].
Another potential mechanism for CD4 down-modulation is internalization and intracellular degradation of CD4–clenoliximab complex. In response to stimuli that activate T cells, CD4 endocytosis has been observed [14]. Thus it is plausible that clenoliximab binding may elicit some degree of CD4 endocytosis. Reduced lymphocyte expression of CD4 is another potential mechanism of CD4 down-modulation. These mechanisms, however, would not explain the presence of sCD4 complexes described in the present report. Potentially, a combination of stripping with internalization and/or reduced expression causes the down-modulation observed in patients administered clenoliximab.
For a significant portion of patients (30%) in the 350 mg weekly and 700 mg alternate-week dose groups, sCD4–clenoliximab complexes were not detected despite maximal down-modulation. Also, the amount of complex detected was variable between patients treated similarly. There was no obvious correlation between patient characteristics and the lack of complex. These observations may have been due to differences in the structure and immunoreactivity of the recombinant sCD4, used as a standard in the immunoassay, relative to the endogenous stripped patient CD4.
Only a small fraction of the total circulating clenoliximab was present within the sCD4 complex (generally less than 2% at Tmax, the time-point at which complex concentration was maximal). However, interestingly, sCD4, in complex with clenoliximab, apparently accumulated to amounts in serum exceeding that of circulating cell-associated CD4. Assuming modulation is caused entirely by stripping, one can estimate the amount of sCD4 that would be generated from circulating T lymphocytes. This estimate can be derived from a patient's baseline number of T lymphocytes (unchanged throughout treatment), a receptor density of 50 000 CD4 molecules per cell [15], the magnitude of the decrease in OKT4 MFI and 1:1 binding stoichiometry.
At complex Tmax for the patient receiving a single dose of 1 mg/kg (Fig. 1), the amount of stripped CD4 in serum was >4 times higher than expected on the basis of the above assumptions. Measured sCD4 in the complex was >10 times the amount of circulating cell-associated CD4 in the blood at Tmax. For the patient receiving a single dose of 10 mg/kg (Fig. 1), the amount of stripped CD4 in serum was 22 times higher. For this patient, measured sCD4 in the complex was >60 times the amount of circulating cell-associated CD4 in the blood at Tmax. These relative concentrations of cell-associated CD4, sCD4–clenoliximab complex and free clenoliximab at complex Tmax are depicted schematically in Fig. 5A.
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We propose that the accumulation of CD4 in the circulation following clenoliximab treatment reflects stripping and down-modulation of CD4 in well-perfused tissues rich in CD4 lymphocytes. Free mAb perfuses tissue rich in T cells, then coats and strips the CD4 from the cell surface. sCD4–clenoliximab complex in tissues subsequently re-equilibrates with the blood compartment. Movement of the T cells, however, from tissue to blood, is largely restricted. This process is depicted schematically in Fig. 5B
. Thus accumulation of sCD4–clenoliximab complex in the blood may reflect the pharmacological activity of the molecule in the lymphoid tissues. In vitro studies suggest that CD4 down-modulation is dependent upon antibody cross-linking of macrophage Fc receptor [6]. Though clenoliximab binds Fc receptor with low affinity, Fc receptors are more highly expressed on tissue-resident macrophages than circulating macrophages. Clenoliximab interaction with tissue macrophage Fc receptor may therefore occur with greater frequency in lymphoid tissue relative to peripheral blood. The pharmacodynamic profile of clenoliximab following multiple i.v. infusion was consistent with that expected on the basis of single-dose data. Using pharmacokinetic/pharmacodynamic modelling and data from the single-dose protocol, multiple-dose pharmacodynamic profiles were simulated to predict patient response following multiple administration, and to optimize the design of multiple-dose trials [8]. The data from this first multiple-dose trial of clenoliximab was consistent with our expectation, on the basis of these simulations, that higher doses administered less frequently would result in pharmacodynamic profiles similar to lower doses administered more frequently.
In summary, single and multiple infusions of clenoliximab cause regimen- and time-dependent coating, stripping and down-modulation of CD4 in patients, without cellular depletion. These activities prevent interaction of cell-associated CD4 with MHC class II molecules on antigen-presenting cells and inhibit T-cell proliferation and cytokine production. Though not yet proven to be related to clinical outcome, these changes in the state of the T cell probably play an important role in the clinical activity of this antibody.
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Acknowledgments |
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The authors wish to thank D. Kwok, W. Shi, E. Minthorn and A. Dang for expert technical assistance and D. Mould, M. Elliott and A. Sharma for many interesting discussions.
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Notes |
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Correspondence to: T. W. Hepburn, GlaxoSmithKline, Department of Drug Metabolism and Pharmacokinetics, 709 Swedeland Road, PO Box 1539 (UW2720), King of Prussia, PA 19406, USA. E-mail: timothy_w_hepburn{at}gsk.com
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