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. 2005 Dec;116(4):487–498. doi: 10.1111/j.1365-2567.2005.02247.x

Construction and characterization of a humanized anti-human CD3 monoclonal antibody 12F6 with effective immunoregulation functions

Bohua Li 1,2, Hao Wang 1,2, Jianxin Dai 1, Junjie Ji 1, Weizhu Qian 1, Dapeng Zhang 1, Sheng Hou 1, Yajun Guo 1,2,3
PMCID: PMC1802437  PMID: 16313362

Abstract

12F6 is a murine anti-human CD3 monoclonal antibody, which competes with OKT3 for binding to human T cells and possesses more effective T-cell suppression and activation properties compared to OKT3. It thus exhibits the potential to be developed as an immunoregulation agent for manipulating T-cell functions and preventing acute allograft rejection. In an attempt to minimize the immunogenicity of murine 12F6 (m12F6) for potential clinical application, a humanized version of 12F6, denoted as hu12F6, was successfully constructed by complementary determining region (CDR) grafting and shown to maintain both T-cell activation and suppression activities similar to m12F6. Furthermore, in order to reduce the first dose reaction syndrome caused by T-cell activation following the first administration of anti-CD3 antibodies, two amino acid mutations were introduced into the Fc region of hu12F6, resulting in the Fc-mutated 12F6 humanized antibody (hu12F6mu). This Fc-mutated version displayed a similar antigen-binding affinity and specificity compared with hu12F6 and m12F6 but with much weaker FcR binding activity. hu12F6mu was shown to be much less potent in the induction of T-cell proliferation, cytokine release (tumour necrosis factor-α, interferon-γ and interleukin-10) and early activation marker expression on the cell surface (CD69 and CD25) than parental 12F6 and OKT3 did. In contrast, hu12F6mu was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with a similarity as compared to m12F6 and OKT3. In conclusion, the resultant hu12F6mu was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to OKT3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.

Keywords: Humanization, CD3, monoclonal antibody, first dose syndrome, immunosuppression

Introduction

A major limitation in the clinical use of murine monoclonal antibodies (mAb) is the induction of human anti-mouse antibody response (HAMA), which causes rapid clearance of injected antibodies and reduced their efficiency.1 Because it is difficult to generate human mAb for clinical use with conventional hybridoma technique, generation of chimeric or humanized antibodies by genetic engineering technology becomes an alternative solution to this major problem. Chimeric antibodies can be constructed by fusing the rodent antigen-binding variable domains to the human constant region of immunoglobulin and usually have a better binding affinity and a reduced immunogenicity compared with their mouse counterparts. However, clinical experiences indicated that some of chimeric antibodies were still highly immunogenic and could elicit a strong anti-antibody reaction when these antibodies were injected into humans. The resulting antibodies were predominantly directed to the idiotype of the antibodies.2–4 To further reduce the immunogenicity of chimeric antibodies, humanized antibodies have been constructed by grafting the CDRs of a murine antibody into the corresponding regions of a human antibody.5,6 Currently, humanized antibodies have showed their significant potentials as therapeutic agents clinically.

OKT3 is a murine anti-human CD3 monoclonal antibody that possesses both T-cell activation and suppression properties. Mouse OKT3 antibody was approved for the reversal of acute kidney transplant rejection in 1986 and subsequently for treatment of cardiac and liver transplant rejection. In spite of its efficacy, the broad use of OKT3 in clinical treatment was severely limited by the HAMA response and first dose reaction. First-dose reaction is a syndrome consisting of fever, chills, dyspnoea, tachycardia, emesis, and diarrhoea following the first and, in some cases, following the second OKT3 administration.7–9 Previous studies indicated that first-dose reactions resulted from T cell activation by OKT3 and concomitant cytokine release. It had been shown that T cell activation by anti-CD3 antibodies involves a complex molecular interaction. Soluble anti-CD3 antibodies were unable to induce the activation of T cells in vitro in the absence of Fc receptor-bearing cells.10–12 Such activation depends on T-cell receptor (TCR)/CD3 complex cross-linking on the surface of T cells, which was mediated by anti-CD3 antibody linking both T cells (via its variable regions) and Fc receptor-bearing cells (via its Fc portion).13,14 Hence, Several forms of anti-CD3 mAb15,16 containing mutations in the upper CH2 region (from positions 234–237) have been constructed and proved to have reduced affinity for Fc receptor. These Fc-mutated anti-CD3 mAbs were shown to be significantly less mitogenic to T cells.

12F6 is a murine anti-human CD3 monoclonal antibody produced by a routine hybridoma technique.17 Previous studies indicated 12F6 competed with OKT3 for binding to T cells and possesses more effective T-cell activation and suppression properties, suggesting that 12F6 might be a better type of immunosuppressive agent. To reduce the immunogenicity and mitogenicity of 12F6 for potential future clinical use, we constructed a humanized 12F6 antibody with a mutated Fc region and studied its biological properties in vitro. This Fc-mutated humanized antibody was shown to retain potent immunosuppression but with a remarkable attenuation in T-cell activation, suggesting that it might become a more effective immunosuppressive agent with less immunogenicity and toxicity for the treatment of transplantation patients.

Materials and methods

Materials

OKT3 mouse hybridoma cells were obtained from The American Type Culture Collection. 12F6 mouse hybridoma cells were kindly provided by Dr JT Wong (Massachusetts General Hospital, Boston, MA). Monoclonal antibodies OKT3 (immunoglobulin G2a (IgG2a), κ) and 12F6 (IgG2a, κ) were purified by Protein A affinity chromatograph from hybridoma cell culture supernatant and antibody concentrations were determined by absorbance at 280 nm. Each purified antibody was labelled with fluoroscein isothiocyanate (FITC) to produce OKT3–FITC or 12F6–FITC. FITC-conjugated anti-CD3 mouse monoclonal antibodies SK7–FITC and UCHT1–FITC were purchased from Becton Dickinson (San Jose, CA) and Beckman Coulter (Miami, FL), respectively. The two vectors,18 pAH4604 and pAG4622 were kindly provided by Pr. SL Morrison (Department of Microbiology and Molecular Genetics, UCLA, Los Angeles, CA). Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation.

Flow cytometry analysis

Flow cytometry analysis (FCM) was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). For one-colour analysis, fluorescence intensity was plotted on the x axis and cell number was plotted on the y axis. For two-colour analysis, green fluorescence (FITC) intensity was plotted on the x axis and red fluorescence (phycoerythrin, PE) intensity was plotted on the y axis.

Cloning of 12F6 heavy and light chain variable region genes

RNA was isolated from 12F6 hybridoma cells with TRIzol Reagent (Gibco BRL, Grand Island, NY). The heavy and light variable region cDNAs of 12F6 were cloned from hybridoma cells using 5′RACE system (Gibco BRL, Gaithersburg, MD) according to the manufacture's instruction. The final polymerase chain reaction (PCR) products were cloned into pGEM-T vector (Promega, Madison, WI) for sequence determination. The gene-specific primers (GSP) for PCR amplification of heavy chain were as follows: GSP1-H, 5′-AGC TGG GAA GGT GTG CAC ACC ACT-3′; GSP2-H, 5′-CAG AGT TCC AGG TCA AGG TCA-3′; GSP3-H, 5′-CTT GAC CAG GCA TCC TAG AGT-3′. The gene-specific primers for PCR amplification of light chain were as follows: GSP1-L,5′-TTG CTG TCC TGA TCA GTC CAA CT-3′; GSP2-L, 5′-TGT CGT TCA CTG CCA TCA ATC TT-3′; GSP3-L,5′-TTG TTC AAG AAG CAC ACG ACT GA-3′.

Construction of chimeric antibody expression vector

Using a PCR method, EcoRV and XbaI sites were added to the 5′ end of the heavy chain variable region gene (VH) and a NheI site added to the 3′ end. The PCR product was cloned into pGEM-T vector and the sequence was verified. The VH was excised by EcoRV and NheI digestion and then, inserted into the EcoRV/NheI sites of the pAH4604 vector containing the human gamma-1 constant region gene (CH). The resultant pAH4604-VH vector was cleaved with XbaI and BamHI and then the 3·3 kb fragment containing chimeric rodent/human antibody heavy chain gene was cloned into the pCDNA3·1(–) vector (Invitrogen, San Diego, CA) which had been digested with the same restriction enzymes, yielding the chimeric heavy chain expression vector pcDNA3·1(–)VHCH. The human kappa chain constant cDNA(CL) was obtained as a 0·3 kb PCR product derived from pAG4622. The light chain variable region gene (VL) of 12F6 was fused to the 5′ end of the CL by overlapping PCR method. The resultant chimeric light chain gene (VLCL) with a HindIII site upstream of the start codon and an EcoRI site downstream of the stop codon was cloned into pGEM-T vector and its sequence was verified. The VLCL was excised by HindIII and EcoRI digestion and ligated into the pcDNA3·1Zeo (+) vector (Invitrogen) cleaved with the same restriction enzymes, yielding the chimeric light chain expression vector pcDNA3·1Zeo (+)VLCL.

Construction of humanized antibody expression vector

The VH of human antibody KOL was chosen as framework for the humanized heavy chain and the VL of human Bence-Jones protein REI was chosen for the humanized light chain. The humanized versions of 12F6 were designed referring to the strategy described by Adair et al.19 The light and heavy chain variable region genes of humanized antibodies were synthesized by overlapping PCR method. The light and heavy chain expression vectors for humanized antibodies were constructed in an identical manner to the chimeric antibody described above.

Construction of Fc-mutated humanized antibody heavy expression vector

Two amino acid mutations (L234A, L235A) in the Fc region of the 12F6 humanized antibody were introduced by overlapping PCR to generate Fc-mutated humanized heavy chain gene. The heavy chain expression vector for Fc-mutated humanized antibody was also constructed in an identical manner to the chimeric heavy chain expression vector described above.

Antigen-binding activity assays

Appropriate light and heavy expression vectors for producing recombinant antibodies were cotransfected into COS-7 cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. After 72 hr incubation, the cell supernatants were collected and analysed by enzyme-linked immunosorbent assay (ELISA) for quantization of recombinant antibodies. The ELISA assay used goat anti-human IgG Fc (KPL, Gaithersburg, MD) as capture antibody and goat anti-human kappa-horseradish peroxidase (HRP) (Southern Biotechnology Associates, Birmingham, AL) as detecting antibody. Purified human IgG1/Kappa (Sigma, Saint Louis, MO) was used as a standard control. FCM analysis was performed to determine the binding of recombinant antibodies to PBMCs. Briefly, human PBMCs at 1 × 106 cells/ml were incubated with various dilutions of the culture supernatants containing recombinant antibodies for 1 hr at 4°. The cells were washed and incubated with FITC-goat anti-human IgG(H + L) (Zymed, San Francisco, CA) for 1 hr at 4°. Then the cells were washed and analysed by FCM.

Stable expression and purification of chimeric, humanized, Fc-mutated humanized antibodies

Appropriate light and heavy expression vectors were cotransfected into Chinese hamster ovary (CHO)-K1 cells using Lipofectamine 2000 reagent. Stable transfectants were isolated by limiting dilution in the presence of 600 µg/ml G418 and 300 µg/ml Zeocin. The culture supernatants from individual cell clone were analysed for antibody production by the sandwich ELISA described above. The clones that produced the highest amount of recombinant antibodies were selected and grown in serum-free medium. The recombinant antibodies were purified by Protein A affinity chromatography from the serum-free culture supernatant. Antibody concentration was determined by absorbance at 280 nm.

Competitive binding assay

Human PBMCs at 1 × 105 cells/well were incubated with subsaturated concentrations of FITC-conjugated antibodies and increasing concentrations of purified competing antibodies for 1 hr at 4°. The cells were counterstained with PE-conjugated anti-human CD5 (BD Biosciences, San Diego, CA) to identify T lymphocytes and then, analysed by two-colour FCM.

FcR binding competition assay

The human cell line U937 bearing both FcRI and FcRII20,21 was used in FcR binding competition assays. Briefly, U937 cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mm l-glutamine and 500 U/ml human interferon-γ (INF-γ) for 24 hr at 37°. The human INF-γ was used to enhance the expression of FcRI on U937 cells.22,23 Then the cells were washed and then incubated with subsaturated concentrations of FITC-conjugated mouse IgG2a,κ antibodies (specific for trintrophenyl (TNP); BD Biosciences) and increasing concentrations of purified anti-CD3 antibodies for 1 hr at 4°. After incubation, the cells were washed and analysed by FCM.

T lymphocyte proliferation assays

Human PBMCs at 1 × 105 cells/well were incubated with serial log dilutions of anti-CD3 antibodies for 3 days at 37°. On the third day, [3H]thymidine was added at 1 µCi/well. After a 16-hr incubation, cells were harvested and [3H]thymidine incorporation was measured in a liquid scintillation counter.

Cytokine release assays

Human PBMCs were incubated at 1 × 106 cells/ml with serial log dilutions of anti-CD3 antibodies at 37°. The supernatants were collected at 24 hr for ELISA assay of tumour necrosis factor-α (TNF-α) (GeneMay, San Diego, CA) or 72 hr for ELISA assays of IFN-γ and interleukin-10 (IL-10) (Endogen, Cambridge, MA).

Detection of the expression of early T-cell activation marker

Human PBMCs were incubated at 1 × 106 cells/ml with serial log dilutions of anti-CD3 antibodies at 37°. After 16 hr incubation, the cells were harvested and stained with FITC-labelled anti-CD69 (BD Biosciences) and PE-conjugated anti-CD5 for CD69 determination. After 36 hr, the cells were harvested and stained with FITC-labelled anti-CD25 (BD Biosciences) and PE-conjugated anti-CD5 (BD Biosciences) for CD25 determination. Finally, the stained cells were analysed by two-colour FCM. PE-conjugated anti-CD5 was used to identify T lymphocytes.

CD3 modulation

To determine the ability of m12F6 to modulate TCR/CD3 complex in vitro, human PBMCs at 1 × 106 cells/ml were incubated for 16 hr at 37° with serial log dilutions of m12F6 or control antibody OKT3. Following incubation, PBMCs were washed and stained with: FITC-goat anti-mouse IgG(H + L) (Zymed, San Francisco, CA) (MC1) or 10 µg/ml OKT3 for 45 min followed by FITC-goat anti-mouse IgG(H + L) (MC2). Stained cells were analysed by FCM. The formula for calculating CD3 modulation was:

graphic file with name imm0116-0487-mu1.jpg

where MC represents the mean channel along the x axis and control cells indicates PBMCs that were not incubated for 16 hr at 37° with anti-CD3 antibodies. mAb-treated cells mean the PBMcs that were incubated for 16 hr at 37° with anti-CD3 antibodies.

To determine the abilities of the chimeric antibody (c12F6), humanized antibody (hu12F6), Fc-mutated humanized antibody (hu12F6mu) to modulate TCR/CD3, humam PBMCs at 1 × 106 cells/ml were incubated for 16 hr at 37° with serial log dilutions of c12F6, hu12F6 or hu12F6mu. Following incubation, PBMCs were washed and stained with: FITC conjugated goat anti-human IgG(H + L)(MC1) or 10 µg/ml c12F6 for 45 min followed by FITC-goat anti-human IgG(H + L)(MC2). Stained cells were analysed by FCM. The formula for calculating CD3 modulation is described above.

Mixed lymphocyte reaction assays

PBMCs from unrelated healthy donors were designated as responder and stimulator cells, respectively. The stimulator PBMCs were exposed to 3000 rad of radiation. Responder PBMCs (2 × 105 cells/well) were cocultured with irradiated stimulator PBMCs (2 × 105 cells/well) in the presence of different concentrations of anti-CD3 antibodies in 96-well microtitre plates at 37°. After a 5-day culture, [3H]thymidine was added at 1 µCi/well. Sixteen hr later, the cells were harvested and [3H]thymidine incorporation was measured in a liquid scintillation counter.

Results

In vitro characterization of 12F6

There are several anti-CD3 monoclonal antibodies, including 12F6, OKT3, SK7 and UCHT1. Competition binding assays indicated that 12F6 competed with OKT3 for binding to human T cells. Both 12F6 and OKT3 were able to effectively inhibit the binding of two other anti-CD3 antibodies SK7 and UCHT1 (Fig. 1). The abilities of 12F6 antibody to activate T cells in vitro as compared with OKT3 were evaluated by T-cell proliferation, cytokine (TNF-α, IFN-γ and IL-10) release and early activation marker (CD69 and CD25) expression assays. Immunosuppressive activity of 12F6 antibody was evaluated by examining its capacity to modulate TCR/CD3 complex and inhibit mixed lymphocyte reaction. These results demonstrated that 12F6 possessed more effective T-cell activation and suppression properties compared to OKT3 (in Figs 6–10) the * denotes a significant difference (P < 0·05 by Student's unpaired t-test) between 12F6 and OKT3).

Figure 1.

Figure 1

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Competition binding assays of anti-CD3 antibodies. Human PBMCs were incubated with fixed concentrations of FITC-conjugated antibodies and increasing concentrations of competitor antibodies (12F6 or OKT3) and negative control antibody CD45 for 1 hr at 4°. The cells were counterstained with PE-conjugated anti-human CD5 and analysed by two-colour FCM. Maximal fluorescence means the mean channel on the x axis obtained in the absence of competitor antibodies. CD45 is a purified mouse anti-human CD45 monoclonal antibody that does not compete with anti-CD3 antibodies. All data were represented as the mean of triplicate samples.

Figure 6.

Figure 6

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T-cell proliferation induced by anti-CD3 antibodies. Human PBMCs at 1 × 105 cells/well were incubated with serial log dilutions of OKT3, m12F6, c12F6, hu12F6, or hu12F6mu. On the third day, [3H]thymidine was added at 1 µCi/well. After a 16-hr incubation, [3H]thymidine incorporation was measured. CD8, a mouse anti-human CD8 monoclonal antibody is a negative control. All data were represented as the mean of triplicate samples. c.p.m., counts per minute.

Figure 10.

Figure 10

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Inhibition of mixed lymphocyte reaction by anti-CD3 antibodies. Responder PBMCs and irradiated stimulator PBMCs from unrelated donors were cocultured for 5 days in the presence of different concentrations of OKT3, m12F6, c12F6, hu12F6, or hu12F6mu. [3H]thymidine was added and [3H]thymidine incorporation was measured after 16 hr. All data were expressed as the mean of triplicate samples. (R +Ri), responder PBMCs mixed with irradiated self PBMCs in the absence of antibody; (R), responder PBMCs alone in the absence of antibody; (S), stimulator PBMCs alone in the absence of antibody; (R +Si), responder PBMCs mixed with irradiated stimulator PBMCs in the absence of antibody.

Construction of chimeric antibody

The variable region cDNAs for the light and heavy chains of m12F6 were cloned from the hybridoma cells by 5′RACE. The nucleotide sequences of VH and VK were determined and deposited in GenBank under the accession numbers AY646725 and AY646726, respectively. The deduced amino acid sequences of VH and VL were shown in Fig. 2. The expression vectors for chimeric light chain and heavy chain were constructed and coexpressed transiently in COS cells to produce a chimeric antibody (c12F6). The c12F6 antibody from COS cell culture medium was tested in an antigen-binding activity assay and shown to bind well to human T cells, which indicated that the light and heavy variable regions of m12F6 were correctly cloned and produced. CHO cells were cotransfected with chimeric light and heavy chain expression vectors, and then stable transfectants were obtained after selection in present of G418 and Zeocin. Finally, c12F6 was purified from the CHO cell serum-free culture supernatant.

Figure 2.

Figure 2

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Amino acid sequences of humanized 12F6 heavy (a) and light (b) chain variable regions. 12F6VH and 12F6VL indicate heavy and light chain variable regions of murine 12F6 antibody, respectively. The heavy chain variable region of human antibody KOL was chosen as framework for the humanized heavy chain and the light chain variable region of human Bence-Jones protein REI was chosen for the humanized light chain. RHa and RHj indicate different versions of humanized heavy chain variable regions. RLa and RLi indicate different versions of humanized light chain variable regions. The dashes represent amino acids that are the same as the corresponding residues in human antibodies KOL or REI. The CDRs are enclosed with brackets. Amino acids (in one-letter notation) are numbered according to Kabat.24

Construction of humanized and Fc-mutated humanized antibodies

Firstly, the three Complementarity-determining regions from 12F6 light chain or heavy chain were directly grafted into human antibody light chain or heavy chain frameworks to generate humanized antibody genes. The humanized VL and VH were cloned into expression vectors, respectively, and were coexpressed transiently in COS cells, yielding humanized version (RLa/RHa). The amino acid sequence of RLa/RHa was shown in Fig. 2. Humanized antibody in COS cell culture supernatant was quantified by ELISA and the binding of RLa/RHa to PBMCs was determined by FCM. The result indicated that this antibody almost lost its binding activity (Fig. 3). This suggested that some human framework region (FR) residues must be altered to reconstitute the full binding activity. The important FR residues that may have influences on binding activity were analysed and a backmutation assay was also performed. A number of humanized light and heavy chain versions were constructed to evaluate the contribution of FR residues to antigen binding activity. Finally, a humanized antibody (RLi/RHj) showing the same antigen binding activity as compared with c12F6 was obtained (Fig. 3). The humanized version (RLi/RHj) was designated as hu12F6 and its amino acid sequences were shown in Fig. 2. To meet the need of further studies, hu12F6 was expressed in CHO cells and the CHO cell transfectants that stably produced antibodies were obtained by a positive selection procedure. In the same way, the hu12F6 light chain expression vector and the Fc-mutated hu12F6 heavy chain expression vector were coexpressed in CHO cells to obtain Fc-mutated humanized antibody (hu12F6mu). Finally, both hu12F6 and hu12F6mu were obtained and purified from the CHO cell serum-free culture supernatant.

Figure 3.

Figure 3

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Antigen binding assays for 12F6 humanized antibodies. Human PBMCs were incubated with serial log dilutions of c12F6, humanized version (RLa, RHa) or humanized version (RLi, RHj) for 1 hr at 4°. Cells were washed and incubated with FITC-goat anti-human IgG (H + l) for 1 hr at 4°æ. Cells were then washed and analysed by FCM. All data were expressed as the mean of triplicate samples.

Competitive binding assay

In the competitive binding assay, c12F6 and hu12F6 effectively competed with m12F6 for binding to T cells, which indicated that these constructed chimeric and humanized antibodies possessed affinity and specificity similar to m12F6 (Fig. 4). Although hu12F6mu had two amino acid mutations on the heavy chain constant region compared with hu12F6, it was also shown to have similar avidity in comparison with m12F6 (Fig. 4).

Figure 4.

Figure 4

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Binding of 12F6-FITC to human PBMCs in the presence of increasing concentrations of competitor antibodies: m12F6, c12F6, hu12F6 or hu12F6mu. Human PBMCs were incubated at 1 × 106 cells/ml with subsaturated concentrations of 12F6-FITC and increasing concentrations of m12F6, c12F6, hu12F6, hu12F6mu or CD45 for 1 hr at 4°. The cells were counterstained with PE-conjugated anti-human CD5 to identify T lymphocytes and then analysed by two-colour FCM. Maximal fluorescence means the mean channel fluorescence on the x axis obtained in the absence of competitor antibodies. CD45 is a mouse anti-human CD45 monoclonal antibody that does not compete with anti-CD3 antibodies. All data were expressed as the mean of triplicate samples.

FcR binding competition assay

The Fc portions of murine IgG2a and human IgG1 antibodies have a high affinity for human FcRI, and they can also bind to the human FcRII.23,25 In this study, the abilities of m12F6, hu12F6 and hu12F6mu to bind to FcR on U937 cells were investigated and compared. As shown in Fig. 5, m12F6, a murine IgG2a antibody, possessed the highest affinity for human FcR on U937 cells. The hu12F6 could effectively compete with m12F6 for binding to FcR, which indicated it also had a high FcR binding affinity. As expected, hu12F6mu, which was designed to eliminate the FcR binding capacity of hu12F6, bound to U937 cells poorly, suggesting it had much weaker FcR binding activity than that of hu12F6.

Figure 5.

Figure 5

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Binding competition of anti-CD3 antibodies with FITC-conjugated mouse IgG2a,κ antibody to human U937 cells. INF-γ-treated human U937 cells at 106 cells/ml were incubated with subsaturated concentrations of FITC-conjugated mouse IgG2a,κ antibodies (specific for TNP) and increasing concentrations of m12F6, hu12F6 or hu12F6mu for 1 hr at 4°. Then the cells were washed and analysed by FCM. Maximal fluorescence means the mean channel fluorescence obtained in the absence of competitor antibodies. All data were expressed as the mean of triplicate samples.

T-cell proliferation induced by anti-CD3 antibodies

T-cell activation by the recombinant anti-CD3 antibodies was often assessed by T-cell proliferation, cytokine release and early T-cell activation marker expression on the T-cell surfaces. First, the three antibodies c12F6, hu12F6 and hu12F6mu, were evaluated for their abilities to induce T-cell proliferation. Because chimeric antibody contained murine variable region, it usually kept the biological activity of murine mAb. As expected, c12F6 was shown to have mitogenic potency comparable to m12F6 (Fig. 6). The abilities of hu12F6 and hu12F6mu to induce mitosis of T cells were also compared with that of m12F6. The results (Fig. 6) showed that the mitogenic potencies of hu12F6 were similar to m12F6 and it could induce marked T-cell proliferation over a wide concentration range (0·1–1000 ng/ml). However, no significant T-cell proliferation was induced by hu12F6mu at concentrations even up to 10 ng/ml. Hu12F6mu could only induce T-cell proliferation at higher antibody concentrations and its mitogenic potency was much weaker than that of m12F6, hu12F6 and control anti-CD3 antibody, OKT3 (Fig. 6).

Release of TNF-α, IFN-γ and IL-10 induced by anti-CD3 antibodies

To further study the T-cell activation by the anti-CD3 antibodies, the release of three cytokines, TNF-α, IFN-γ and IL-10, was examined. As illustrated in Fig. 7, both c12F6 and hu12F6 induced the secretion of cytokines TNF-α, IFN-γ and IL-10 comparably to m12F6. The release of these cytokines was detectable with the three mAb at each concentration used in this experiment. In contrast, hu12F6mu could not induce the secretion of IFN-γ at each used concentration and the release of TNF-α and IL-10 was only detectable with hu12F6mu at a concentrations of 10 ng/ml or higher. Moreover, the secretion levels of TNF-α and IL-10 induced by hu12F6mu were much lower than m12F6 and control antibody OKT3 at the same antibody concentrations (Fig. 7).

Figure 7.

Figure 7

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Release of TNF-α, IFN-γ and IL-10 induced by anti-CD3 antibodies. Human PBMCs were incubated with serial log dilutions of OKT3, m12F6, c12F6, hu12F6 or hu12F6mu. The supernatants were collected at 24 hr for ELISA assay of TNF-α or at 72 hr for ELISA assays of IFN-γ and IL-10. CD8 is a negative control antibody. All data were represented as the mean of triplicate samples.

Expression of early T-cell activation markers on cells treated with the antibodies

Activation of T cells can be monitored by measuring the expression levels of early activation markers, especially for CD69 and CD25. The abilities of c12F6, hu12F6 and hu12F6mu to induce expression of these markers were investigated in this experiment (Fig. 8). It was demonstrated that c12F6 and hu12F6 possessed potency in inducing CD69 and CD25 expression equivalent to that of m12F6. However, hu12F6mu was shown to be much less potent in the induction the two early activation markers as compared with m12F6 and control antibody OKT3.

Figure 8.

Figure 8

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Expression of CD69 and CD25 induced by anti-CD3 antibodies. Human PBMCs were incubated with serial log dilutions of OKT3, m12F6, c12F6, hu12F6 or hu12F6mu. The cells were harvested at 16 hr for CD69 determination or at 36 hr for CD25 determination. CD8 is a negative control antibody. All data were represented as the mean of triplicate samples.

Immunosuppressive properties of anti-CD3 antibodies

Immunosuppressive activities of anti-CD3 antibodies were evaluated by determining their abilities to modulate TCR/CD3 complex and inhibit mixed lymphocyte reaction. As shown in Fig. 9, c12F6, hu12F6 and hu12F6mu could effectively modulate the TCR/CD3 complex on the treated T cells. Ninety percent TCR modulation could be achieved in presence of each of the three antibodies at a concentration of 1000 ng/ml. It was demonstrated that the capacities of these three antibodies to modulate TCR/CD3 complex were similar to that of m12F6 (Fig. 9). In the mixed lymphocyte reaction assays, all of the anti-CD3 antibodies could inhibit lymphocyte proliferation compared to no antibody control (Fig. 10). Chimeric, humanized and Fc-mutated humanized versions of m12F6 antibody displayed similar capacities in suppressing mixed lymphocyte reaction compared with the parental mouse antibody, 12F6 (Fig. 10). These results suggested that c12F6, hu12F6 and hu12F6mu conserved potent immunosuppressive activities equivalent to that of murine 12F6.

Figure 9.

Figure 9

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Comparison of the abilities of anti-CD3 antibodies to modulate the TCR/CD3 complex. CD3 modulation were quantified by FCM as described in Methods. All data were expressed as the mean of triplicate samples.

Discussion

To reduce the immunogenicity of murine mAb, humanized antibodies have been developed during the past 10 years and have been proved to be highly effective therapeutics. In this paper, we have described the successful humanization of 12F6, a murine anti-human CD3 monoclonal antibody. 12F6 could effectively inhibit the binding of OKT3 to T cells and possess more effective T-cell activation and suppression properties compared with OKT3. To further investigate its potential as an immunosuppressive drug for transplantation patients, humanization of 12F6 was performed to reduce its immunogenicity. The humanized version (RLa/RHa) was constructed by simply grafting CDRs from 12F6 light or heavy chain to human antibody light or heavy chain. This version almost lost all the binding activity. It is not surprising, because, in most cases, the successful design of high affinity CDR-grafted antibodies requires that key murine FR residues be substituted into the human acceptor framework to preserve the CDR conformations.26–30 To reconstitute the full binding activity, a number of light and heavy chain versions were produced to evaluate the contribution of FR residues to antigen binding activity. It was demonstrated that residues 6, 23, 24, 27, 28, 30, 71, 48, 49, 73, 76 and 78 on the heavy chain framework regions (FRs) and residues 21, 46, 47 and 73 on the light chain FRs have important effects on antigen-binding activities of humanized antibodies. Backmutation of all these residues generated the humanized version (RLi/RHj), which completely restored the binding avidity of the parental mouse antibody.

Besides HAMA, the clinical use of anti-CD3 antibodies has been severely limited by another obstacle, first-dose reactions. Previous studies indicated that first-dose reactions resulted from T-cell activation and cytokine release following the administration of anti-CD3 antibodies. Cytokines, such as TNF-α, IFN-γ, IL-2, IL-6 and granulocyte–macrophage colony-stimulating factor, were believed to be mainly responsible for the acute toxicity of OKT3.4,8,31,32 In an attempt to generate anti-CD3 mAb with less toxicity, several forms of anti-CD3 mAb were developed. Because this activation was triggered by anti-CD3 mAb cross-linking T cells and Fc receptor-bearing cells,13,14 the anti-CD3 F(ab′)2 fragments, which lacked the Fc portion and could not bind to Fc receptor-bearing cells, demonstrated a significant reduction in T-cell activation with retention of immunosuppression. But the use of anti-CD3 F(ab′)2 fragments in clinical treatment is limited by several drawbacks: (1) it is difficult to produce abundant rigorously purified F(ab′)2 fragments; (2) the minimal contamination of anti-CD3 F(ab′)2 fragments by whole mAb results in significant retention of mitogenic potency; (3) more importantly, because of the short serum half-life of F(ab′)2 fragments, repeated administrations are necessary to achieve efficacy.33,34 In conclusion, the anti-CD3 F(ab′)2 fragment form is not suitable for clinical treatment Therefore, efforts are made to construct whole anti-CD3 mAb molecule with less affinity for Fc receptor by genetic engineering. These mutated anti-CD3 mAb were shown to be significant less mitogenic to T cells.15

In this study, T-cell activation properties of recombinant 12F6 antibodies were assessed by T-cell proliferation, cytokine release and early T-cell activation marker expression. Three cytokines, TNF-α, IFN-γ and IL-10, were selectively measured. TNF-α and IFN-γ, which belong to T helper 1 (Th1) type cytokines are partially responsible for adverse side-effects of anti-CD3 mAb,31,32 whereas IL-10, a Th2 type cytokine, is a potent inhibitor of the release of TNF-α and IFN-γ induced by anti-CD3 mAb. Previous studies are also supportive of a major role of IL-10 in the down-modulation of the anti-CD3 antibody-triggered T-cell activation cascade.35,36 The expression levels of early T-cell activation markers CD69 and CD25 by anti-CD3 mAb were also determined in this experiment. CD69 is the earliest T-cell surface activation marker. It can be detected at the cell surface within a few hours after the stimulation of TCR/CD3 and its peak expression was achieved by 18–24 hr. Once expressed, CD69 acts as a costimulatory molecule for T-cell activation and proliferation.37–40 CD25, the α chain of high-affinity IL-2 receptors, is not expressed on resting T cells, but it can be rapidly induced to express on cell surface after T-cell activation.41–43 Our results showed both c12F6 and hu12F6 had T-cell activation properties similar to native m12F6. It could be deduced that when these two recombinant antibodies were used in human therapy, the first-dose reaction associated with T-cell activation by anti-CD3 antibody would still happen. In order to develop a humanized anti-CD3 antibody with as less acute toxicity in human as possible, we introduced two amino acid mutations (L234A, L235A) into the Fc region of hu12F6, resulting in the Fc-mutated 12F6 humanized antibody, namely hu12F6mu. These results demonstrated that the FcR binding affinity of hu12F6mu was much less than that of hu12F6 and suggested the ability of the antibody to bind to Fc receptor-bearing cells was greatly impaired. More importantly, hu12F6mu had similar antigen-binding avidity compared with hu12F6, indicating that these changes on Fc region had no influence the antibody's binding to CD3 antigen. In the T-cell activation assays, hu12F6mu was shown to be much less potent in inducing T-cell proliferation, cytokine release (both Th1 and Th2 cytokines) and early activation marker expression than hu12F6, m12F6 and control antibody OKT3 did. This suggested that hu12F6mu might have a remarkable reduction on first-dose reaction when used in humans clinically.

Immunosuppressive properties of anti-CD3 antibodies in vivo may have been a consequence, at least in part, of antigenic modulation of TCR/CD3 complex, with consequent loss of the ability of T cells to recognize their target antigens.44–47 Most of anti-CD3 mAb including OKT3 and 12F6 possess potent T-cell suppression property, which makes them potential candidates for immunosuppressive agents. In this study, immunosuppressive activities of anti-CD3 antibodies were evaluated by testing their abilities to modulate the TCR/CD3 complex and inhibit mixed lymphocyte reaction in vitro. hu12F6mu, a 12F6 humanized antibody bearing two amino acid mutations on the Fc region, showed similar capability to modulate CD3 antigen and inhibit mixed lymphocyte reaction in comparison with hu12F6 and m12F6. This suggested the immunosuppressive property of this humanized antibody was not altered by the amino acid changes on its constant region.

In summary, the 12F6 humanized antibody (hu12F6) has been successfully constructed and demonstrated to possess T-cell activation and suppression properties equivalent to m12F6. hu12F6mu is a mutant derived from hu12F6 by genetic modification on the Fc region. This Fc mutant of hu12F6 has been prepared to reduce the first dose reaction elicited by T-cell activation, which severely limits the clinical use of the anti-CD3 antibodies including the approved drug product OKT3. hu12F6mu was shown to be significantly less potent in T-cell activation but maintained the important immunosuppressive ability. Thus, hu12F6mu is likely to be much less immunogenic and toxic to patients than OKT3. In conclusion, hu12F6mu, the Fc-mutated anti-CD3 humanized antibody, might have the potential to become a novel immunosuppressive agent for the treatment of acute organ transplantation rejection with better safety profiles and greater efficacy than the OKT3 mouse antibody.

Acknowledgments

This work was supported by grants from Shanghai Commission of Science & Technology, Ministry of Science & technology of China (973 program project), National Natural Science Foundation of China and a special financial support from Shanghai E-Institute, Immunology Division and Pudong Bureau of Science & Technology. The authors thank Ms. Yang Yang, Ms. Jing Xu and Ms. Xiaoyuan Wang for their excellent technical assistance.

Abbreviations

CDR

complementary-determining region

CH

human gamma-1 constant region gene

CL

human kappa chain constant cDNA

FCM

flow cytometry

FRs

framework regions

GSP

gene-specific primers

HAMA

human anti-mouse antibody response

mAb

monoclonal antibodies

PBMCs

peripheral blood mononuclear cells

VH

heavy chain variable region gene

VL

light chain variable region gene

References

  • 1.Vaswani SK, Hamilton RG. Humanized antibodies as potential therapeutic drugs. Ann Allergy Asthma Immunol. 1998;81:105–19. doi: 10.1016/S1081-1206(10)62794-9. [DOI] [PubMed] [Google Scholar]
  • 2.Jaffers GJ, Fuller TC, Cosimi AB, Russell PS, Winn HJ, Colvin RB. Monoclonal antibody therapy. Anti-idiotypic and non-anti-idiotypic antibodies to OKT3 arising despite intense immunosuppression. Transplantation. 1986;41:572–8. doi: 10.1097/00007890-198605000-00004. [DOI] [PubMed] [Google Scholar]
  • 3.Hesse CJ, Heyse P, Stolk BJ, Balk AH, Jutte NH, Mochtar B, Weimar W. The incidence and quantity of antiidiotypic antibody formation after OKT3 monoclonal therapy in heart-transplant recipients. Transplant Proc. 1990;22:1772–3. [PubMed] [Google Scholar]
  • 4.Norman DJ. Mechanisms of action and overview of OKT3. Ther Drug Monit. 1995;17:615–20. doi: 10.1097/00007691-199512000-00012. [DOI] [PubMed] [Google Scholar]
  • 5.Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986;321:522–5. doi: 10.1038/321522a0. [DOI] [PubMed] [Google Scholar]
  • 6.Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature. 1988;332:323–7. doi: 10.1038/332323a0. [DOI] [PubMed] [Google Scholar]
  • 7.Woodle ES, Thistlethwaite JR, Jolliffe LK, Fucello AJ, Stuart FP, Bluestone JA. Anti-CD3 monoclonal antibody therapy. An approach toward optimization by in vitro analysis of new anti-CD3 antibodies. Transplantation. 1991;52:361–8. [PubMed] [Google Scholar]
  • 8.Gaston RS, Deierhoi MH, Patterson T, et al. OKT3 first–dose reaction: association with T cell subsets and cytokine release. Kidney Int. 1991;39:141–8. doi: 10.1038/ki.1991.18. [DOI] [PubMed] [Google Scholar]
  • 9.Thistlethwaite JR, Stuart JK, Mayes JT, Gaber AO, Woodle S, Buckingham MR, Stuart FP. Complications and monitoring of OKT3 therapy. Am J Kidney Dis. 1988;11:112–9. doi: 10.1016/s0272-6386(88)80192-6. [DOI] [PubMed] [Google Scholar]
  • 10.Landegren U, Andersson J, Wigzell H. Mechanism of T lymphocyte activation by OKT3 antibodies. A general model for T cell induction. Eur J Immunol. 1984;14:325–8. doi: 10.1002/eji.1830140409. [DOI] [PubMed] [Google Scholar]
  • 11.Looney RJ, Abraham GN. The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3. J Immunol. 1984;133:154–6. [PubMed] [Google Scholar]
  • 12.Ceuppens JL, Meurs L, Van Wauwe JP. Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function. J Immunol. 1985;134:1498–502. [PubMed] [Google Scholar]
  • 13.Palacios R. Mechanisms by which accessory cells contribute in growth of resting T lymphocytes initiated by OKT3 antibody. Eur J Immunol. 1985;15:645–51. doi: 10.1002/eji.1830150702. [DOI] [PubMed] [Google Scholar]
  • 14.Ceuppens JL, Bloemmen FJ, Van Wauwe JP. T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex. J Immunol. 1985;135:3882–6. [PubMed] [Google Scholar]
  • 15.Cole MS, Anasetti C, Tso JY. Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells. J Immunol. 1997;159:3613–21. [PubMed] [Google Scholar]
  • 16.Xu D, Alegre ML, Varga SS, et al. In vitro characterization of five humanized OKT3 effector function variant antibodies. Cell Immunol. 2000;200:16–26. doi: 10.1006/cimm.2000.1617. [DOI] [PubMed] [Google Scholar]
  • 17.Wong JT, Colvin RB. Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens. J Immunol. 1987;139:1369–74. [PubMed] [Google Scholar]
  • 18.Coloma MJ, Hastings A, Wims LA, Morrison SL. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Meth. 1992;152:89–104. doi: 10.1016/0022-1759(92)90092-8. [DOI] [PubMed] [Google Scholar]
  • 19.Adair JR, Athwal DS, Bodmer MW, et al. Humanization of the murine anti-human CD3 monoclonal antibody OKT3. Hum Antibodies Hybridomas. 1994;5:41–7. [PubMed] [Google Scholar]
  • 20.Looney RJ, Abraham GN, Anderson CL. Human monocytes and U937 cells bear two distinct Fc receptors for IgG. J Immunol. 1986;136:1641–7. [PubMed] [Google Scholar]
  • 21.Jones DH, Looney RJ, Anderson CL. Two distinct classes of IgG Fc receptors on a human monocyte line (U937) defined by differences in binding of murine IgG subclasses at low ionic strength. J Immunol. 1985;135:3348–53. [PubMed] [Google Scholar]
  • 22.Perussia B, Dayton ET, Lazarus R, Fanning V, Trinchieri G. Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells. J Exp Med. 1983;158:1092–113. doi: 10.1084/jem.158.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Alegre ML, Collins AM, Pulito VL, et al. Effect of a single amino acid mutation on the activating and immunosuppressive properties of a ‘humanized’ OKT3 monoclonal antibody. J Immunol. 1992;148:3461–8. [PubMed] [Google Scholar]
  • 24.Kabat EA, Wu TT, Reid-Miller M, Perry HM, Gottesman KS. Sequences of proteins of immunological interest. 4. Washington DC: United States Department of Health and Human Services; 1987. [Google Scholar]
  • 25.Duncan AR, Woof JM, Partridge LJ, Burton DR, Winter G. Localization of the binding site for the human high-affinity Fc receptor on IgG. Nature. 1988;332:563–4. doi: 10.1038/332563a0. [DOI] [PubMed] [Google Scholar]
  • 26.Kipriyanov SM, Little M. Generation of recombinant antibodies. Mol Biotechnol. 1999;12:173–201. doi: 10.1385/MB:12:2:173. [DOI] [PubMed] [Google Scholar]
  • 27.Queen C, Schneider WP, Selick HE, et al. A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci USA. 1989;86:10029–33. doi: 10.1073/pnas.86.24.10029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ohtomo T, Tsuchiya M, Sato K, et al. Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions. Mol Immunol. 1995;32:407–16. doi: 10.1016/0161-5890(95)00017-9. [DOI] [PubMed] [Google Scholar]
  • 29.Carter P, Presta L, Gorman CM, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA. 1992;89:4285–9. doi: 10.1073/pnas.89.10.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pulito VL, Roberts VA, Adair JR, et al. Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A. J Immunol. 1996;156:2840–50. [PubMed] [Google Scholar]
  • 31.Abramowicz D, Schandene L, Goldman M, et al. Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation. 1989;47:606–8. doi: 10.1097/00007890-198904000-00008. [DOI] [PubMed] [Google Scholar]
  • 32.Chatenoud L, Ferran C, Legendre C, et al. In vivo cell activation following OKT3 administration. Systemic cytokine release and modulation by corticosteroids. Transplantation. 1990;49:697–702. doi: 10.1097/00007890-199004000-00009. [DOI] [PubMed] [Google Scholar]
  • 33.Woodle ES, Thistlethwaite JR, Ghobrial IA, Jolliffe LK, Stuart FP, Bluestone JA. OKT3 F(ab′)2 fragments – retention of the immunosuppressive properties of whole antibody with marked reduction in T cell activation and lymphokine release. Transplantation. 1991;52:354–60. doi: 10.1097/00007890-199108000-00033. [DOI] [PubMed] [Google Scholar]
  • 34.Hirsch R, Bluestone JA, DeNenno L, Gress RE. Anti-CD3 F(ab′)2 fragments are immunosuppressive in vivo without evoking either the strong humoral response or morbidity associated with whole mAb. Transplantation. 1990;49:1117–23. doi: 10.1097/00007890-199006000-00018. [DOI] [PubMed] [Google Scholar]
  • 35.Schandene L, Gerard C, Crusiaux A, Abramowicz D, Velu T, Goldman M. Interleukin-10 inhibits OKT3-induced cytokine release: in vitro comparison with pentoxifylline. Transplant Proc. 1993;25(2 Suppl. 1):55–6. [PubMed] [Google Scholar]
  • 36.Herbelin A, Abramowicz D, de Groote D, Naret C, Kreis H, Bach JF, Goldman M, Chatenoud L. CD3 antibody-induced IL-10 in renal allograft recipients: an in vivo and in vitro analysis. Transplantation. 1999;68:616–22. doi: 10.1097/00007890-199909150-00004. [DOI] [PubMed] [Google Scholar]
  • 37.Risso A, Smilovich D, Capra MC, Baldissarro I, Yan G, Bargellesi A, Cosulich ME. CD69 in resting and activated T lymphocytes. Its association with a GTP binding protein and biochemical requirements for its expression. J Immunol. 1991;146:4105–14. [PubMed] [Google Scholar]
  • 38.Bajorath J, Aruffo A. Molecular model of the extracellular lectin-like domain in CD69. J Biol Chem. 1994;269:32457–63. [PubMed] [Google Scholar]
  • 39.Testi R, Phillips JH, Lanier LL. Leu 23 induction as an early marker of functional CD3/T cell antigen receptor triggering. Requirement for receptor cross-linking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase C. J Immunol. 1989;142:1854–60. [PubMed] [Google Scholar]
  • 40.Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells. 1994;12:456–65. doi: 10.1002/stem.5530120502. [DOI] [PubMed] [Google Scholar]
  • 41.Biselli R, Matricardi PM, D'Amelio R, Fattorossi A. Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Scand J Immunol. 1992;35:439–47. doi: 10.1111/j.1365-3083.1992.tb02879.x. [DOI] [PubMed] [Google Scholar]
  • 42.Depper JM, Leonard WJ, Kronke M, Noguchi PD, Cunningham RE, Waldmann TA, Greene WC. Regulation of interleukin 2 receptor expression: effects of phorbol diester, phospholipase C, and reexposure to lectin or antigen. J Immunol. 1984;133:3054–61. [PubMed] [Google Scholar]
  • 43.Morris JC, Waldmann TA. Advances in interleukin 2 receptor targeted treatment. Ann Rheum Dis. 2000;59(Suppl. 1):i109–i114. doi: 10.1136/ard.59.suppl_1.i109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hirsch R, Eckhaus M, Auchincloss H, Sachs DH, Bluestone JA. Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses. J Immunol. 1988;140:3766–72. [PubMed] [Google Scholar]
  • 45.Landegren U, Ramstedt U, Axberg I, Ullberg M, Jondal M, Wigzell H. Selective inhibition of human T cell cytotoxicity at levels of target recognition or initiation of lysis by monoclonal OKT3 and Leu-2a antibodies. J Exp Med. 1982;155:1579–84. doi: 10.1084/jem.155.5.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Van Seventer GA, Kuijpers KC, van Lier RA, de Groot ER, Aarden LA, Melief CJ. Mechanism of inhibition and induction of cytolytic activity in cytotoxic T lymphocytes by CD3 monoclonal antibodies. J Immunol. 1987;139:2545–50. [PubMed] [Google Scholar]
  • 47.Anasetti C, Martin PJ, Storb R, et al. Treatment of acute graft-versus-host disease with a nonmitogenic anti-CD3 monoclonal antibody. Transplantation. 1992;54:844–51. doi: 10.1097/00007890-199211000-00015. [DOI] [PubMed] [Google Scholar]

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