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CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Type 1 diabetes (T1D) is a prototypic organ-specific autoimmune disease resulting from the selective destruction of insulin-secreting β-cells within the pancreatic islets of Langerhans. It is caused by an immune-mediated inflammation, involving autoreactive CD4⁺ and CD8⁺ T lymphocytes that infiltrate the islets and initiate insulitis. The use of exogenous insulin is the current standard treatment. However, in spite of significant advances, this therapy is still associated with major constraints, including risk of hypoglycemia and severe degenerative complications. As T1D mainly affects children and young adults, any candidate immune therapy must be safe, and it must avoid a sustained depression of immune responses with all its attendant problems of recurrent infection and drug toxicity. In this context, inducing or restoring immune tolerance to target autoantigens would be the ideal approach. We refer to immune tolerance here as the selective damping of the damaging autoimmune response following a short treatment, while keeping intact the capacity of the host to respond normally to exogenous antigens. The therapeutic approach we discuss in this article originates from attempts to induce tolerance both to soluble antigens and tissue antigens (i.e. alloantigens and autoantigens) by using biological agents that selectively interfere with lymphocyte activation, namely polyclonal and monoclonal anti-T cell antibodies. The challenged dogma was that, in an adult-primed immune system, it was not possible to restore self-tolerance therapeutically without the use of exogenous autoantigen administration. The reality has been that, in diabetes, endogenous host autoantigen can fulfill this role because a significant amount of functioning β-cells remains, even at the time of established hyperglycemia. Experimental results obtained in the 1990s showed that a short-term CD3 antibody treatment in recently diagnosed diabetic non-obese diabetic (NOD) mice induced permanent remission of the disease by restoring self-tolerance. Based on these findings, phase I, II, and III trials were conducted using two distinct humanized Fc-mutated antibodies to human CD3, namely ChAglyCD3 (otelixizumab) and OKT3γ1 Ala-Ala (teplizumab). Overall, when dosing was adequate, the results demonstrated that CD3 antibodies preserved β-cell function very efficiently, maintaining significantly high levels of endogenous insulin secretion in treated patients for up to 24 months after treatment. These data provided the first proof of concept for a long-term therapeutic effect in T1D following a short course administration of a therapeutic agent. Our aim is to review these data and to discuss them in the context of the pitfalls linked to pharmaceutical development, especially in the context of pediatric patients, as in autoimmune diabetes.

Despite remarkable progress in organ transplantation through the development of a wealth of immunosuppressive drugs highly effective at controlling acute rejection, two major problems still remain, the loss of transplants due to chronic rejection and the growing number of sensitized recipients due to previous transplants, transfusions or pregnancies. Induction of immune tolerance appears to be the only way to curb this complex situation. Here we describe that a therapy, already successfully used to restore immune tolerance to self-antigens in overt autoimmunity, is effective at promoting transplant tolerance. We demonstrate that a short low-dose course with CD3 antibodies started after transplantation, at the time of effector T cell priming to alloantigens, induces permanent acceptance of fully mismatched islet allografts. Mechanistic studies revealed that antigen-specific regulatory and effector T cells are differentially affected by the treatment. CD3 antibody treatment preferentially induces apoptosis of activated alloreactive T cells which is mandatory for tolerance induction. In contrast, regulatory T cells are relatively spared from CD3 antibody-induced depletion and can transfer antigen-specific tolerance thus arguing for their prominent role in sustaining long-term graft survival., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Monoclonal antibodies have proven to be potent agents to promote immunological tolerance in animal models of autoimmune disease and transplantation. However, optimal clinical application and pharmaceutical development have been limited by the species specificity of therapeutic antibodies, as well exemplified in the case of anti-CD3 antibodies. Compelling evidence in the nonobese diabetic (NOD) mouse, recently translated to clinical autoimmune insulin-dependent diabetes, demonstrates that a short CD3 antibody treatment effectively and durably controls disease progression. We established transgenic mice expressing the human ε chain of the CD3 complex bred onto the NOD background. These mice developed a high incidence of spontaneous autoimmune diabetes and harbored T cells sensitive both in vitro and in vivo to anti-human CD3 antibodies. Treatment of diabetic transgenic mice with otelixizumab, an anti-human CD3 antibody that has proven effective in the clinic, resulted in durable disease remission dependent on transferable T cell-mediated tolerance. This model should enable the evaluation of anti-human CD3 antibodies to determine their potential clinical utility.

Purpose of Review: The continuing problem of late graft loss and immunosuppressive drug toxicity forces us to explore new treatments for the induction of transplant tolerance. Monoclonal antibodies targeting molecules implicated in lymphocyte activation, in particular CD3/TCR, constitute a promising strategy., Recent Findings: Promising results were obtained from the use of antibodies targeting CD3/TCR, coreceptors or costimulatory pathways as tolerance-promoting tools in experimental transplantation. These antibodies do not uniformly depress the immune system but act in an antigen-specific manner by preferentially targeting effector T cells while preserving regulatory T cells. However, translation to the clinic proved to be more difficult than expected. New generation CD3 antibodies, currently used in phase II/III trials in autoimmunity, constitute a promising approach as, beside their immunosuppressive effect, they also express potent tolerogenic capacities. Importantly, CD3 therapy is effective especially when applied in primed hosts, highlighting the importance of the therapeutic window for tolerance induction., Summary: Further investigations are required for adapting to the clinic monoclonal antibodies as substitutes for current immunosuppression. Our aim is to show that development of new therapeutic strategies/molecules may come from transversal-type research, in particular from experience in autoimmunity, as immune responses leading to autoimmunity and graft rejection involve similar pathways.

Otelixizumab is a chimeric CD3 antibody that has been genetically engineered to remove the glycosylation site in the Fc domain. This limits its ability to bind to complement or Fc receptors and reduces the risk of adverse clinical reactions due to cytokine release. In a trial for treatment of type 1 diabetes, a short treatment with otelixizumab resulted in a reduced requirement for insulin lasting at least 18 months. In the course of this trial, the blood concentrations of the antibody were measured by flow cytometry to determine its pharmacokinetic profile. Dose-dependent accumulation of otelixizumab was demonstrated and modeling of the data indicated that the terminal half-life was approximately 1.5 days. Antibody responses to otelixizumab were measured by 2 methods: a bridging enzyme-linked immunosorbent assay and surface plasmon resonance. The surface plasmon resonance method had a greater sensitivity and was able to detect responses in all patients, starting at 8 days after the commencement of therapy. Neutralizing antibodies were detected in a significant proportion of patients by days 22 to 29. Although no adverse clinical effects were associated with these antibody responses and they did not appear to affect the clearance of the drug, they might have important implications for possible retreatment of the patients.

Type 1 diabetes mellitus (T1DM) is a prototypic organ-specific autoimmune disease that results from selective destruction of insulin-secreting beta-cells by immune-mediated inflammation (insulitis), that is, the infiltration of pancreatic islets by autoreactive CD4(+) and CD8(+) T lymphocytes. Current treatment is substitutive-chronic use of exogenous insulin-which, in spite of considerable advances, is still associated with constraints and lack of effectiveness over the long-term in relation to the prevention of vascular and neurological complications. Finding a cure for T1DM is an important medical health challenge, as the disease's incidence is steadily increasing in industrialized countries and projections of future prevalence are alarming. Crucially, as T1DM mainly affects children and young adults, any candidate immune therapy must be safe and avoid chronic use of immunosuppressants that promote sustained depression of immune responses. The ideal approach would, therefore, involve induction or, in the case of established T1DM, restoration of immune tolerance to target autoantigens. This Review presents, in particular, two strategies that are still in clinical development but hold great promise. These strategies are focused on the use of candidate autoantigens and anti-CD3 monoclonal antibodies.

Here we report a unique situation in which an early and synchronized Epstein-Barr virus (EBV) reactivation was induced by a 6-day course of treatment with a humanized CD3-specific monoclonal antibody in patients with recent onset of type 1 diabetes. The virologic and immunologic analysis demonstrated that this reactivation was transient, self-limited, and isolated, associated with the rapid advent of an EBV-specific T-cell response. The anti-CD3 antibody administration induced short-lasting immunosuppression and minor yet clear-cut signs of T-cell activation that preceded viral reactivation. Early posttransplant monitoring of renal and islet allograft recipients showed that no comparable phenomenon was observed after the administration of full-dose immunosuppressive therapy. This EBV reactivation remains of no apparent clinical concern over the long term and should not preclude further development of therapeutic anti-CD3 antibodies. This phenomenon may also direct new research avenues to understand the still ill-defined nature of stimuli triggering EBV reactivation in vivo.

Purpose of Review: A major problem in the field of clinical transplantation, as well as in autoimmunity, is that conventional treatments rely on chronic immunosuppression that is not specific for the antigens involved and that increases the risk of infections and tumours. A major need and challenge is, therefore, to achieve 'operational tolerance', namely an inhibition of pathogenic responses in the absence of chronic immunosuppression., Recent Findings: Here we review data showing that monoclonal antibodies to the CD3 complex, the signal transducing element of the T cell receptor, promote immune tolerance. This strategy has been the matter of extensive experimental studies in models of autoimmunity and has recently led to a successful clinical translation., Summary: Results from controlled trials in autoimmune insulin-dependent diabetes showed that CD3 monoclonal antibodies afford long-term effects following a short administration. The present challenge is to build on these results, first, to set the use of CD3 monoclonal antibodies as an established therapy in well selected subsets of patients with autoimmunity, and second, given the similarities of immune mechanisms underlying T cell-mediated autoimmune diseases and allograft rejection, to address if and how this therapeutic strategy could be extended to organ transplantation in the not-too-distant future.

Aim: Infliximab (IFX) therapy is highly efficacious for the induction and maintenance of remission in pediatric Crohn disease (CD). However, to date it is unclear how long patients should be given IFX. Given the increasing safety concerns about the concomitant and prolonged use of IFX and azathioprine in CD, we wanted to address the clinical outcome in pediatric CD patients who responded to IFX medication, once IFX was stopped., Patients and Methods: Upon induction therapy with 3 IFX infusions, 36 of 38 patients with CD were in clinical remission at 3 months. These 36 responders were separated into 2 groups: 16 patients received no further IFX infusions, whereas 20 patients received scheduled maintenance therapy with IFX for 12 months., Results: Among the 16 patients who received no further IFX infusions, 12 experienced relapse during the 12-month follow-up interval after IFX was stopped. In the group receiving maintenance therapy, 11 of 20 patients remained in clinical remission at 12 months of therapy, whereas 8 patients required adjustment of IFX doses or intervals. Among the 11 children who were in clinical remission and receiving maintenance therapy without dose adjustment, 8 experienced relapse within 12 months after IFX maintenance therapy was stopped. Overall, the relapse rates after IFX induction or maintenance therapy was stopped were 75% and 72%, respectively., Conclusions: These data indicate that IFX is efficacious in controlling severe pediatric CD; however, to induce and maintain clinical remission, repeated IFX infusions are required, with a need for dose adjustment in a substantial number of patients.

CD3-specific monoclonal antibodies were the first rodent monoclonals introduced in clinical practice in the mid 1980s as approved immunosuppressants to prevent and treat organ allograft rejection. Since then compelling evidence has been accumulated to suggest that in addition to their immunosuppressive properties, CD3-specific antibodies can also afford inducing immune tolerance especially in the context of ongoing immune responses. Thus, they are highly effective at restoring self-tolerance in overt autoimmunity, a capacity first demonstrated in the experimental setting, which was recently transferred to the clinic with success.

In developed countries, autoimmune diseases represent the third major cause of morbidity and mortality after cancer and atherosclerosis and their incidence has steadily increased over the past three decades. Conventional therapeutic approaches are essentially palliative, anti-inflammatory or immunosuppressive; in addition, they are non-specific, unrelated to the antigens involved in disease pathogenesis. This explains the growing attention to modern technologies that made new biological agents and methods available. A few of these are already approved for regular clinical practice; others are still in clinical development but hold great promise. The question is: will these new tools allow us to develop a real cure for autoimmunity, restoring self-tolerance to target autoantigens? This goal is ambitious, namely harnessing the pathogenic immune response while preserving the host response to exogenous or unrelated antigens.

B-cell posttransplantation lymphoproliferative disorder (B-PTLD) is a rare but severe complication of transplantation, with no consensus on best treatment practice. This prospective trial, the first to test a treatment for PTLD, was designed to evaluate the efficacy and safety of rituximab in patients with B-PTLD after solid organ transplantation (SOT). Forty-six patients were included and 43 patients were analyzed. Patients were eligible if they had untreated B-PTLD that was not responding to tapering of immunosuppression. Treatment consisted of 4 weekly injections of rituximab at 375 mg/m2. At day (d) 80, 37 (86%) patients were alive, and the response rate was 44.2%, including 12 complete response/unconfirmed complete response (CR/CRu). The only factor predictive of a response at d80 was a normal lactate dehydrogenase level (P = .007, odds ratio [OR] = 6.9). At d360, responses were maintained in 68% of patients, and 56% of patients were alive. The overall survival rate at 1 year was 67%. We conclude that rituximab is effective and safe in PTLD, with stable responses at 1 year. The response rate and overall survival might be improved by combining rituximab with other treatments.

Tumor necrosis factor (TNF)-alpha plays a critical role in the initiation and progression of Crohn's disease, a chronic inflammatory disorder of the gastrointestinal tract. Infliximab, a chimeric monoclonal antibody blocking TNF-alpha, has proven effective as an induction and maintenance therapy for refractory Crohn's disease in adult and pediatric patients. However, infliximab therapy induces the appearance of neutralizing anti-infliximab antibodies. In the pediatric cohort, we analyzed (n=28) sensitization occurred in 35.7% patients and was associated with a loss of response to maintenance infusions. In two patients presenting high titers of anti-infliximab antibodies, severe infusion reactions were observed, possibly IgE-mediated, precluding further use of the medication. Serum concentrations of TNF-alpha and infliximab were influenced by the presence of anti-infliximab antibodies. We propose that surveillance of circulating infliximab and/or TNF-alpha concentration during maintenance therapy represents an indirect but reliable method to monitor anti-infliximab immunization.

The treatment of autoimmune diseases using conventional chemical immunosuppressants has short-term effects, imposing the need for chronic treatment with its risks of over-immunosuppression. CD3-specific monoclonal antibodies can restore self-tolerance in a durable fashion after a single short-term treatment, as demonstrated in several experimental models and clinically in recent-onset insulin-dependent diabetes. Disease remission involves first an immediate 'freezing' of the autoimmune response, which is linked to CD3-specific antibody-induced antigenic modulation of CD3-TCR complex at the T lymphocyte surface, followed by 'resetting' of TGF-beta-dependent T-cell mediated immunoregulation. Tolerance induction is demonstrated by persisting disease protection in spite of recovery of full immunocompetence to unrelated antigens.

Over the last few years, there has been a re-emergence of the concept of suppressor/regulatory T cells among the central players of immune mechanisms controlling a wide variety of immune responses from physiological autoreactivity (i.e., response to self-antigens) to responses to transplants, tumours and infectious antigens. Regulatory T cells are diverse in their phenotypes, antigen specificity, mode of action and immunopathological relevance. This review briefly summarises studies from the authors' group showing that specialised subsets of regulatory T cells are instrumental in the control of autoimmune diseases and more specifically of Type 1 diabetes. In addition, this review will provide evidence supporting the notion that CD3-specific monoclonal antibodies are representatives of a new category of immunotherapeutic agents that possess the unique capacity to promote immunological tolerance (an antigen-specific unresponsiveness in the absence of long-term generalised immunosuppression) through their ability to induce immunoregulatory T cells.

In vivo administration of anti-CD3 Ab induces both immune tolerance and undesirable side-effects resulting from nonspecific proinflammatory cytokine production. In the current study, we investigated the therapeutic potential of two structurally altered forms of the anti-CD3 Ab in ameliorating established experimental autoimmune encephalomyelitis. Administration of either a chimeric (NM-IgG3) or digestion product (NM-F(ab')2) form of the anti-CD3 Ab during established experimental autoimmune encephalomyelitis conferred significant protection from clinical disease progression and was associated with decreased Ag-specific T cell proliferation, cytokine production, and CNS inflammation. Interestingly, while this protection correlated with an increase in the frequency of CD4(+)CD25(+) regulatory T cells, neither prior depletion of regulatory T cells nor anti-TGF-beta treatment abrogated the treatment's efficacy. Importantly, both treatments induced normal levels of intracellular Ca(2+)-flux, but significantly diminished levels of TCR signaling. Consequent to this decreased level of TCR-mediated signaling were alterations in the level of apoptosis and CD4+ T cell trafficking resulting in a profound lymphopenia. Collectively, these results indicate that nonmitogenic anti-CD3 directly induces a state of immune unresponsiveness in primed pathogenic autoreactive effector cells via mechanisms that may involve the induction of T cell tolerance, apoptosis, and/or alterations in cell trafficking.

Monoclonal antibodies are homogeneous sets of immunoglobulins with well-defined specificity and biochemical characteristics. They were introduced into clinical practice in the early 1980s, and since then their use has rapidly expanded. Most of the side effects observed with first-generation murine (mouse or rat) antibodies have been successfully overcome with the advent of humanized (chimeric or CDR-grafted) and more recently fully human antibodies. Our aim is to review the major steps in the development of therapeutic monoclonal antibodies in the fields of transplantation and autoimmunity, and discuss the salient features of antibodies presently used in the clinic--notably anti-T-cell and anticytokine antibodies. The discussion will also focus on the unique capacity of some monoclonal antibodies to induce immune tolerance.

Current therapeutic approaches in transplantation and autoimmunity are essentially focused on immunosuppression, which is non-specific (i.e. unrelated to the antigens involved). The major drawback is their relative ineffectiveness in the long term, with the likely risk of recurrence of the pathogenic immune process once the drug is withdrawn necessitating indefinite drug administration; this has attendant problems of recurrent infections and drug toxicity. Instead, CD3-specific monoclonal antibodies possess the unique capacity to induce immunological tolerance: an antigen-specific unresponsiveness in the absence of long-term generalised immunosuppression, as is well-established in experimental models. Clinical application using humanised non-mitogenic CD3-specific antibodies is presently underway. The future challenge will be to define the modalities allowing the widespread application of this strategy through a better understanding of the underlying immune mechanisms.

The author hopes to convince the reader that the data presented argue for a stage during the development of IDDM when beta-cell destruction can be counteracted and tolerance to beta cells restored, provided the immune aggression is arrested. This argument constitutes a solid rationale for immunointervention in established IDDM, especially by using potent agents such as CD3. The future for the application of monoclonal antibodies not only in autoimmunity but also in transplantation is exiting. With the development of humanized monoclonal antibodies, therapeutic uses for them are likely to expand. Enormous progress has been made in the last 15 years, and it is likely that before a similar time period has elapsed, monoclonal antibodies will have become standard tools that will dispense the need for long-term immunosuppression and its inherent dangers in various clinical arenas.

Treatment with nonmitogenic CD3 Ab reverses established autoimmune diabetes in nonobese diabetic mice by restoring self-tolerance, and is currently under clinical evaluation in patients presenting recent onset type I diabetes. Due to the immunosuppressive potential of this strategy, it was relevant to explore how this treatment would influence the outcome of concomitant viral infections. In this study, we used a transgenic model of virally induced autoimmune diabetes (rat insulin promoter-lymphocytic choriomeningitis virus) that allows for more precise tracking of the autoaggressive response and choice of the time point for initiation of autoimmunity. CD3 was most effective during a clearly defined prediabetic phase and prevented up to 100% of diabetes by drastically lowering activation of autoaggressive CD8 lymphocytes and their production of inflammatory cytokines. Interestingly, reversion of established disease could be achieved as well, when nonmitogenic CD3 was administered late during pathogenesis to overtly diabetic recipients. Most importantly, competence to clear viral infections was maintained. Thus, administration of nonmitogenic CD3 prevents diabetes by sufficient systemic reduction of (auto)aggressive lymphocytes, but without compromising antiviral immune competence.

The presence of activated CD4(+) T lymphocytes in psoriatic skin plaques suggests an immune-mediated pathogenesis for the disease. Six patients with recalcitrant plaque psoriasis (PASI>12) received a humanized non-depleting monoclonal antibody to CD4 (ORTHOCLONE OKT(R)cdr4a). The antibody was well tolerated. Four weeks from treatment, the mean decrease in PASI score was 46%. In three patients disease remission was prolonged for up to 6 months and, in one case, up to 1 year post-treatment. In all patients, circulating CD4+ T-cell counts remained normal and peripheral OKTcdr4a-coated CD4+ lymphocytes were detected up to 10 days after antibody infusion. These results point to the relevance of CD4+ lymphocytes in psoriasis. They also emphasize that depletion of CD4+ cells is not mandatory to achieve therapeutic effectiveness., (Copyright 1998 Academic Press Limited.)

The immune mechanisms underlying autoimmune diseases and allograft rejection are similar, and T cells play a major role in both processes. Current therapeutic strategies rely on the application of nonspecific immunosuppression using chemicals such as corticosteroids, azathioprine, methotrexate, cyclophosphamide and cyclosporin. One major drawback of these drugs is their relative ineffectiveness over the long term; withdrawal leads to recurrence of the disease, but chronic administration puts patients at risk of being overimmunosuppressed, which can result in an increased incidence of infections and tumors. This review describes the advantages of developing biological agents that target immune receptors on T cells. In some cases, these agents can induce a state of durable, antigen-specific unresponsiveness in the absence of generalized immunosuppression, which could be useful in transplantation and autoimmunity.

Low doses of the CD3 mAb 145 2C11 restored self tolerance to beta cell Ags in adult overtly diabetic NOD mice. Within 2 to 4 wk after treatment, complete and permanent remission of diabetes was observed. Autoreactive T cells were not deleted in CD3 Ab-protected animals as evidenced first, by the persistence of peripheral insulitis and, second, by the capacity of spleen cells from CD3 Ab-treated mice to transfer diabetes to adult irradiated syngeneic recipients. Moreover, the conferred tolerance was reproducibly reversed by a single injection of cyclophosphamide. For 5 to 7 wk after treatment, IFN-gamma production by stimulated spleen cells was significantly decreased in treated animals. One unique feature was that the CD3 Ab-induced tolerance ensued only from treatment of overtly diabetic NOD mice. Durable protection was exclusively observed when treating mice with recent onset disease (14-20 wk old). At variance with this finding, treatment of 4- and 8-wk-old mice was without effect, and complete but transient protection followed the treatment of 12-wk-old NOD mice. The tolerogenic properties of 145 2C11 did not depend on its mitogenic capacity, since nonmitogenic F(ab')2 fragments also appeared potent at inducing durable remission in overtly diabetic NOD, although nonmitogenic CD3 F(ab')2 fragments could mediate T cell signaling, as evidenced by cytokine gene transcription (IL-2, IFN-gamma, IL-4, and IL-10) assessed by PCR on splenocytes from treated mice. A concomitant cyclosporine treatment abrogated the CD3 mAb-induced protection, further pointing to the crucial role of T cell signaling in the effect observed.

Anti-CD3 MoAb treatment is widely used as an immunosuppressive therapy. In the present study we examined the in vitro T cell response in mice having received 24 h before a single i.v. injection of 10 microgram of anti-CD3 MoAb. We found that splenocytes from these mice displayed a dramatically decreased proliferative response to the T cell mitogens concanavalin A (Con A), anti-CD3, phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) + calcium ionophore, while the effect of lipopolysaccharide (LPS) was not impaired. T cell suppression persisted for about 10 days after anti-CD3 injection, returning to normal within 15 days. The F(ab')2 fragment of anti-CD3 had no such effect, indicating the requirement for in vivo activation. At the dose used, anti-CD3 resulted neither in T cell depletion nor in down-modulation of the CD3/T cell receptor (TCR) complex. The low proliferation was also not explained by apoptosis, following secondary challenge with Con A. Splenocytes from anti-CD3-injected mice were highly responsive to IL-2, but generated little or no IL-2, IL-3, IL-4 and interferon-gamma (IFN-gamma) when exposed to Con A. Normal cytokine production could not be restored by the addition of optimal doses of IL-2 during Con A stimulation. Transforming growth factor-beta (TGF-beta) was the only cytokine whose mRNA expression was not modified in stimulated splenocytes from anti-CD3-injected mice. Furthermore, anti-TGF-beta antibodies increased Con A-induced T cell proliferation, but not cytokine production.

Administration of monoclonal antibodies to CD3 triggers acute and massive release of several cytokines, including tumor necrosis factor alpha (TNF-alpha), essentially T cell-derived. This cytokine release is responsible for the spontaneously reversible acute clinical syndrome observed in most OKT3-treated patients. We found that the first OKT3 injection in human renal allograft recipients led to the release in significant amounts of soluble TNF receptors (TNF-sR55 and TNF-sR75) that are considered main natural inhibitors of TNF bioactivity. As for OKT3-induced TNF-alpha, peak TNF-sR levels were observed 1 hr postinjection, and this release was limited to the first monoclonal antibody injection. A distinct regulation of OKT3-mediated release of TNF-sR75 and TNF-sR55 was observed, since (1) in clear contrast with OKT3-mediated TNF-sR75 induction, TNF-sR55 release was completely blocked by a high dose of corticosteroids prior to OKT3 injection and (2) secretion of TNF-sR75 but not TNF-sR55 correlated with immunoreactive TNF-alpha release. In hemodialyzed patients prior to transplantation and OKT3 treatment, a condition characterized by chronic TNF-alpha release, TNF-sR efficiently block TNF bioactivity. In contrast, the system is overwhelmed by the massive acute TNF-alpha release that follows the first OKT3 injection: in such a condition TNF-sR looses its capacity to counteract TNF bioactivity.

CD3 antibodies are proven immunosuppressants capable of reversing transplant rejection episodes. Their general application has been limited both by their immunogenicity and, in particular, by the "first-dose" cytokine-release syndrome experienced by patients after the initial administration of antibody. We have produced a set of variants of the humanized YTH 12.5 CD3 monoclonal antibody (mAb) (Routledge et al., Eur. J. Immunol. 1991. 21: 2717) bearing different human heavy (H) chain constant regions, with the intention of finding a form of the antibody that is not able to activate T cells. Comparison of the variants having gamma 1, gamma 2, gamma 3 and gamma 4 H chains in a competitive binding assay showed that antibody avidity was not affected by IgG subclass. Using a sensitive indicator of FcR binding activity (the capacity of the CD3 mAb to redirect cytotoxic T cells to kill the monocytic cell line U-937) we demonstrated a functional hierarchy of gamma 1 = gamma 4 > alpha 2 =/> gamma 3 mb >> gamma 2. An aglycosyl version of the gamma 1 CD3 mAb, produced by site-directed mutagenesis (Asn297 to Ala), still had considerable activity in this assay (intermediate to the gamma 1 and alpha 2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental gamma 1 form. When we tested their ability to stimulate T cell proliferation in vitro in the presence of 5% human serum, all of the wild-type immunoglobulin isotypes were found to be active, although there were T cell donor-dependent variations in the extent of the responses. The aglycosyl gamma 1 mAb was, however, completely non-mitogenic in all of ten donors tested, unless the assay was performed in IgG-free medium. Despite being non-stimulatory, this mAb was also able to inhibit the mixed lymphocyte reaction responses of both naive and primed T cells. Comparison of the gamma 1 and aglycosyl gamma 1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the gamma 1 constant region reduced the in vivo tumor necrosis factor-alpha response by a factor of at least 16-fold. These data suggest that the aglycosyl gamma 1 CD3 mAb is a promising candidate for immunosuppressive therapy without "first dose" side effects.

We have used an antihuman tumor necrosis factor monoclonal antibody, CB006 (murine IgG1), to prevent the OKT3-induced acute clinical syndrome. This syndrome is due to the massive, although transient release in the circulation of various cytokines (TNF, interferon gamma, interleukin 2, interleukin 6) and represents one important side effect linked to in vivo use of OKT3. Fourteen kidney allograft recipients undergoing prophylactic OKT3 therapy were treated with CB006 in a single i.v. injection of either 0.4 mg/kg (group I, 7 patients) or 2 mg/kg (group II, 7 patients), 1 hr before the first OKT3 administration. Nineteen consecutive patients formed a historical control group. None of the CB006-pretreated patients showed any of the common, severe OKT3-associated symptoms (hypotension, respiratory distress, or neurotoxicity), which were observed in 10% of the historical controls. In addition, CB006-treated patients showed a lower frequency of pyrexia (> or = 39 degrees C) and gastrointestinal symptoms. None of the CB006-treated patients presented severe vomiting or diarrhea, defined as repeated episodes inducing significant fluid and electrolyte loss. Two out of the 7 patients in group I and group II had mild transitory diarrhea. Mild single vomiting episodes occurred in 2 group I patients and 3 group II patients. At variance in all controls, gastrointestinal symptoms were long lasting and associated with major prostration due to electrolyte and fluid loss. Importantly, CB006-treated patients who presented mild symptoms had detectable bioactive circulating TNF, showing incomplete inactivation of OKT3-induced TNF by CB006. CB006 was perfectly well tolerated, did not induce xenosensitization, and did not affect the biological or clinical effectiveness of OKT3.

Anti-CD3 monoclonal antibodies are potent immunosuppressants widely used in clinical transplantation to prevent or treat acute allograft rejection. We have used a hamster monoclonal antibody (145 2C11) specific for the epsilon chain of the murine CD3 complex to treat autoimmune insulin-dependent diabetes appearing spontaneously by 15 to 30 weeks of age in female Non Obese Diabetic (NOD) mice. Mice showing overt disease (glycosuria and glycemia > or = 4 g/l) were randomized in two groups receiving either anti-CD3 (5 micrograms/day i.v. for 5 consecutive days) or an identical dose of hamster polyclonal immunoglobulins. Progressive remission of disease was observed, 3 to 6 weeks after the end of treatment, in 80% of anti-CD3 treated mice as compared to 6% of mice in the control group. This remission was maintained long term namely, during the 4 to 5 months (after the end of treatment) observation period. These results open interesting perspectives on the possibility to treat recently diagnosed diabetic patients with therapy showing long term efficacy and no chronic toxicity.

This study analyzed the capacity of an anti-human tumour necrosis factor (TNF) monoclonal antibody, CB006 (murine IgG1, Celltech), to prevent the OKT3-induced acute clinical syndrome. Fourteen renal allograft recipients undergoing prophylactic OKT3 therapy were included. CB006 was administered as a single i.v. injection, 0.4 mg/kg (Group I, 7 patients) and 2 mg/kg (Group II, 7 patients), one hour prior to the first OKT3 administration. Nineteen consecutive patients that were part of a randomized multicenter trial constituted the historical control group. In all patients CB006 was perfectly well tolerated and significantly decreased the frequency of the common OKT3-associated acute symptoms. None of the CB006 pretreated patients showed severe life threatening symptoms (hypotension, respiratory distress or neurotoxicity), observed in 10 per cent of historical controls. At variance with controls, in CB006 treated patients gastrointestinal symptoms (vomiting, diarrhea) and pyrexia (body temperature greater than or equal to 39 degrees C) appeared in low frequency, were mild and short lasting, never promoting major prostration of the patients due to electrolytes and fluid loss. Importantly, the presence in some patients of these mild symptoms, correlated with detectable bioactive TNF in the circulation thus reflecting incomplete blockade by CB006 of OKT3-induced TNF. CB006 pharmacokinetics data further stressed the need for adequate dosage adaptation. CB006 did not affect the biological or clinical effectiveness of OKT3. None of the patients showed evidence of anti-CB006 xeno-sensitization.

A phase I/II trial of the anti-CD4 monoclonal antibody (mAb) was undertaken in seven rheumatoid arthritis patients in order, (1) to investigate changes in clinical symptoms and possible side effects, and (2) to study the pharmacokinetics and to determine the dose required to achieve saturation of antibody binding sites on blood leucocytes. BL4mAb is a murine IgG2a which binds to the group 2B epitope of the V1 N terminal domain of the CD4 molecule. It inhibits syncitium formation by human immunodeficiency virus-infected cells. BL4 was administered by one hour-long intravenous infusion each day, for 10 days. Doses were steadily increased from 20 mg/d to 40 mg/d in the first three patients (group I) in an attempt to reach a serum antibody residual level sufficient to saturate CD4+ circulating cells. The three other patients (group II) received a dose of 40 mg/d during 10 consecutive days. One patient who presented chills and mild fever during the first BL4 infusion was not included in the analysis. No clinical side effects were observed in the six other BL4-treated patients. Clinical parameters of disease activity were improved within the first 14 days. Clinical improvement was still significant at day 30 in five patients, but at day 60, only the Ritchie index was still below pretreatment levels. Delayed type hypersensitivity reactions decreased in the three patients who exhibited positive reactions before BL4 administration. A transient drop in peripheral blood CD4+ lymphocyte counts occurred during each infusion in the first days of treatment. Pre-infusion CD4+ lymphocyte counts were moderately decreased within the first 8 days, but rose to pretreatment levels 3 days after the last infusion. BL4 residual levels in serum steadily increased to reach 8.0 micrograms/ml in group I and 9.8 micrograms/ml in group II. Saturation of BL4 binding sites was achieved after 2 days of treatment in all patients of group II but in only one of group I. Four out of six patients produced antibodies against the anti-CD4 mAb. Immunization appeared between days 12 and 50. This study shows that saturation of anti-CD4 mAb binding sites can be achieved by infusions of high doses (40 mg/d) of BL4 without clinical side effects. The results would encourage further placebo-controlled trials, since no definite conclusion can be drawn from the present study as regards clinical efficacy.

The data presented extend to a larger series of 27 consecutive renal allograft recipients treated prophylactically with OKT3 our previous observation that the acute OKT3-induced clinical syndrome is related to massive release in the circulation of some cytokines, among which are tumor necrosis factor and interferon gamma. In addition, a pilot randomized study was set up including 12 consecutive patients receiving high-dose corticosteroid treatment (0.5 g solumedrol) either before or at the same time as the first OKT3 injection. Results confirm that when corticosteroids are given in sufficient amount and, importantly, 1 hr before the first OKT3 injection, they significantly decrease the release of both tumor necrosis factor and interferon gamma. In addition, the pretreatment with corticosteroids may totally abolish the IL-2 release induced by OKT3. Given the key role the massive although transient cytokine release plays in determining the OKT3-induced acute syndrome, these results provide the biological basis supporting a precise kinetics of administration of high-dose corticosteroids to better decrease the severity of the clinical reaction.