- Email: [email protected]
Anti-CD3 antibodies: towards clinical antigen-specific immunomodulation
Anti-CD3 antibodies: towards clinical antigen-specific immunomodulation Lucienne Chatenoud 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ineffectiveness in the long term, with the likely risk of recurrence of the pathogenic immune process once the drug is withdrawn necessitating indefinite drug administration; this has attendant problems of recurrent infections and drug toxicity.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. Addresses INSERM U580 IRNEM, Hoˆpital Necker 161 Rue de Se`vres, 75015 Paris, France e-mail: [email protected]
Current Opinion in Pharmacology 2004, 4:403–407 This review comes from a themed issue on Immunomodulation Edited by Marc Feldmann and Peter Taylor Available online 19th June 2004 1471-4892/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2004.03.012 Abbreviations IL interleukin TCR T cell receptor TGF transforming growth factor
tive cytotoxic effectors) [2]. On this sole basis in the early 1980s, OKT3 was the first monoclonal antibody that entered clinical practice in transplantation. This was four years before its molecular target, the e chain of the CD3 molecule linked to the T cell receptor (TCR) for antigen recognition, was discovered. The strong immunosuppressive potency of OKT3 was evident from the first clinical trials [3–5], and explained the rapid expansion of its use and its approval by regulatory authorities worldwide by 1984. It is the aim of this brief review to discuss the salient features that have marked the clinical development of CD3-specific antibodies over the past 25 years and the uniqueness of their therapeutic properties. Interestingly, and at variance to what was initially thought, this goes far beyond the field of non-specific immunosuppression to also encompass that of immune tolerance.
Biological properties of CD3-specific antibodies All CD3-specific monoclonal antibodies that have been used in experimental and clinical settings are specific to the e chain of the CD3-specific complex. The CD3 complex is a set of five distinct non-polymorphic polypeptide chains expressed on the T cell surface in association with the TCR for antigen recognition. The chains of the CD3 complex are termed e, z, g and d. The z chain is present as a disulfide-bonded homodimer (z–z) or heterodimer. This heterodimer associates the z chain to Z (an alternate splicing product of the z gene) in mouse cells, and to the g chain of the FceRI and the FcgRIII (CD16) receptors in human cells [6]. The intracellular domains of CD3 polypeptides present immunoreceptor tyrosine-based activation motifs (i.e. one for each g, d and e chain, and three for each z chain) that are involved in signal transduction. At the T cell surface, there are at least two ab heterodimers per TCR/CD3 functional complex ([ab]2[g/de]2[zz]4) [6].
Introduction In the mid 1970s, G Kohler and C Milstein reported the technique of producing murine hybridoma-secreting monoclonal antibodies — homogenous sets of immunoglobulins identical in their physicochemical characteristics and specificity — that won the authors the Nobel prize [1]. A few years later, a monoclonal antibody named OKT3 was produced, recognizing a molecule expressed by all mature human T cells. OKT3 proved highly effective at inhibiting lymphocyte functional capacity (i.e. proliferative responses to transplantation antigens [e.g. alloantigens], as well as the generation of alloreacwww.sciencedirect.com
Mitogenicity In contrast to their potent capacity to inhibit in vitro and in vivo antigen-specific immune responses, CD3-specific antibodies are also endowed with potent mitogenic properties owing to their specificity to the TCR-transducing element. In vitro, this mitogenic potential drives T cell proliferation and cytokine production [7]. In vivo, it promotes what was considered the major side effect linked to murine CD3-specific antibody therapy, namely an acute and severe (although self-limited) ‘flu-like’ syndrome comprising high fever, chills, headache and Current Opinion in Pharmacology 2004, 4:403–407
404 Immunomodulation
gastrointestinal symptoms (i.e. vomiting and diarrhea) from the very first hours following initial CD3-specific antibody administration [8,9]. The syndrome is linked to a massive, although transient, systemic release of several cytokines (i.e. tumour necrosis factor, interferong, interleukin [IL]-2, IL-3, IL-6, IL-10, granulocyte–macrophage colony-stimulating factor and IL-4) [8–12]. The mitogenic capacity of CD3-specific antibodies is monocyte dependent, relying upon the capacity of the Fc antibody portion to interact with Fc receptors on monocyte/macrophages, thereby enhancing cross-linking. Thus, in vitro and in vivo, the mitogenic response varies depending upon the isotype of the murine CD3-specific antibody (IgG2a > IgG1 > IgG2b > IgA); CD3-specific F(ab0 )2 fragments, which lack the Fc portion, are nonmitogenic [13–20]. The fact that non-mitogenic CD3specific antibodies fully retained their therapeutic activity constituted the rationale leading to the design of humanised CD3-specific antibodies with engineered non-Fc receptor binding constant portions [21,22]. It is important to emphasize at this point that non-mitogenic CD3specific antibodies are still able to trigger partial signaling both in vitro and in vivo that is essential to mediate their therapeutic activity [16,23,24].
Modulation Unexpectedly, the in vivo administration of CD3-specific antibodies only led to partial depletion of CD3þ cells. In the spleen and lymph nodes of mice treated with Fc receptor binding CD3-specific antibody (145 2C11), approximately 50% of CD3þ cells were eliminated [25]. The pattern is misleading if one only concentrates on peripheral blood, as is the case in patients, because 30– 60 min after the first OKT3 injection, all T cells disappear [26,27]. This effect is mostly caused by the mitogenic and cytokine-releasing effect mentioned above, which favours cell redistribution within the various compartments, trapping by the reticuloendothelial system (opsonisation) and activation of endothelial cells, leading to increased adhesiveness and lymphocyte marginalisation. Interestingly, mechanisms mediating CD3-specific antibody-induced depletion do not only depend upon the functional capacity of the constant Fc antibody portion (i.e. complement-mediated depletion and antibodymediated cell-mediated cytotoxicity), but also upon the antibody’s fine specificity, leading to redirected T cell lysis upon bridging cytotoxic T cells to the target [28] and the induction of apoptosis (i.e. programmed cell death, to which activated cells appear particularly sensitive) [29]. This explains why some degree of CD3þ T cell depletion is also observed with non-Fc binding CD3-specific antibodies (approximately 20–30% in the spleen and lymph nodes) [14,16]. Remnant (non-depleted) T cells undergo antigenic modulation of CD3/TCR. Thus, binding of the CD3-specific antibody to its receptor promotes microaggregation of the Current Opinion in Pharmacology 2004, 4:403–407
complex, capping and subsequent internalisation or shedding [26,30]. These cells are easily detected as they stain CD3–TCR–CD4þ or CD3–TCR–CD8þ [26,27,30]. Antigenic modulation reverses within a few hours (8–12 h) once the antibody is cleared from the environment [26]. CD3-specific antibody-modulated cells are fully unresponsive to antigen-specific or mitogen stimulation [26].
From murine to humanised CD3-specific antibodies The advent of humanised CD3-specific antibodies was an essential milestone in their clinical development. In addition to decreasing the incidence of sensitization, humanisation of CD3-specific antibodies offered the additional advantage of being able to derive non-mitogenic antibodies that were devoid of the deleterious cytokine-releasing activity but still retained their therapeutic potency [21,22]. Two humanised complementarity determining regiongrafted non-mitogenic CD3-specific antibodies are presently being used in clinical trials. OKT3g1 Ala-Ala was characterised by the group of JA Bluestone and is the humanised non-mitogenic version of OKT3 [22]. It presents two mutations in the Fc human g1 region that lead to a 100-fold decrease in the binding of human Fc receptors when compared with OKT3. It is not mitogenic in vitro and does not promote a significant cytokine release in severe combined immunodeficient mice reconstituted with human splenocytes (hu-SPL-SCID mice) [22]. The second antibody, ChAglyCD3, is derived from the rat YTH 12.5 antibody and was characterised by the group of H Waldmann [21]. It expresses a g1 constant region lacking the CH2 domain glycosylation site. As previously shown for other aglycosylated antibodies, ChAglyCD3 is unable to bind Fc receptors, to activate complement, or to induce T cell proliferation in the presence of human serum [21]. In addition, when injected into mice expressing the human CD3-e chain on T cells as a transgene, ChAglyCD3 induced a significantly reduced release of tumour necrosis factor when compared to the parental antibody [21].
From immunosuppression to tolerance induction OKT3 was first used in clinical practice as an immunosuppressant in association with other conventional chemicals such as corticosteroids, azathioprine and, later, cyclosporin A. As such, it proved extremely potent in both preventing and reversing rejection of various organ allografts, and has been widely used, as is illustrated by over 3000 manuscripts published on the topic. By the mid 1990s, regimens using antibodies devoid of the acute side effects described above became available (i.e. CD25 monoclonal antibodies), which explains why OKT3 was progressively abandoned. www.sciencedirect.com
Anti-CD3 antibodies: towards clinical antigen-specific immunomodulation Chatenoud 405
The two main factors explaining the new wave of interest in the use of CD3-specific antibodies concern both the availability of humanised non-mitogenic CD3-specific monoclonals and the compelling experimental evidence arguing for their unique capacity to promote, in both transplantation and autoimmunity settings, a state of antigen-specific unresponsiveness (i.e. immune tolerance). Experiments performed in a rat transplant model by Hall and colleagues [31,32] first showed that short treatment with a non-mitogenic CD3-specific antibody induced permanent engraftment of fully mismatched vascularized heart grafts. In these rats, as proof of the alloantigenspecific tolerance elicited, secondary donor-matched skin grafts survived indefinitely, compared with third party skin allografts that were normally rejected. In the early post-transplant period, CD3-specific antibody-treated recipients showed a typical Th2 immune deviation as reflected by increased IL-4 and IL-5 mRNA levels in the spleen [32]. Spontaneous autoimmune insulin-dependent diabetes (mediated by autoreactive CD4þ and CD8þ T cells) is observed in non-obese diabetic NOD mice. Ten years ago, our group reported the first data showing that a short five-day low-dose treatment of CD3-specific antibodies could restore self-tolerance to b cell antigens in NOD mice at the time of established disease, thus
inducing long-lasting disease remission without need for insulin treatment [16,17,25]. The effect was specific to b cell autoantigens. Thus, syngeneic islet grafts (normally destroyed within a few days in untreated overtly diabetic NOD females owing to recurrence of the autoimmune attack) survived long-term in mice showing CD3-specific antibody-induced remission that were, however, fully capable of normally rejecting skin allografts [17]. The data collected indicate that the immune mechanisms mediating the tolerogenic capacity of CD3-specific antibodies evolve in two phases: induction and maintenance [25]. The induction phase coincides with CD3-specific antibody administration and is associated with clearing of insulitis and a rapid return to normoglycemia. A transient Th2 polarisation is also observed during this first phase, which appears, however, irrelevant to eliciting the longterm antigen-specific effect, as CD3-specific antibodyinduced long-lasting disease remission was also observed in IL-4 invalidated NOD mice (NOD IL-4/) [25,33]. The maintenance phase is characterised by a state of immunological ‘active’ or ‘dominant’ tolerance involving specialized subsets of regulatory or suppressor T cells. Thus protected mice harbour both diabetogenic T cells and CD4þCD25þ and CD4þCD62Lþ regulatory T cells, as assessed by their capacity to block disease transfer into
Table 1 Clinical use of humanised non-mitogenic CD3-specific antibodies. Clinical indication
Monoclonal antibody
Trial
Number of patients
Results
References
Acute renal allograft rejection
OKT3g1 Ala-Ala treatment for 12 consecutive days (in association with conventional immunosuppressants) ChAglyCD3 treatment for eight consecutive days (in association with conventional immunosuppressants) OKT3g1 Ala-Ala treatment for 14 consecutive days
Phase I
Seven patients on antibody treatment
Good safety profile Reversal of rejection in five out of seven patients
[37]
Phase I
Nine patients on antibody treatment
Good safety profile Reversal of rejection in seven out of nine patients
[36]
Phase I/II
24 (12 active treatment; 12 untreated controls)
[38]
OKT3g1 Ala-Ala treatment for 12–14 consecutive days OKT3g1 Ala-Ala treatment for 12 consecutive days (in association with conventional immunosuppressants) ChAglyCD3 treatment for six consecutive days
Phase I/II
Seven patients on antibody treatment
Phase I
Six patients on antibody treatment
At one year, preservation of b cell mass in nine out of 12 treated patients versus two out of 12 untreated controls Six out of seven patients had 75% improvement in number of inflamed joints Four out of six patients achieved and maintained insulin independence with normal metabolic control
Acute renal allograft rejection
Autoimmune insulindependent diabetes
Psoriatic arthritis
Prevention of islet allograft rejection
Autoimmune insulindependent diabetes
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Phase II 80 (40 active (double blind, treatment; 40 placebo) placebo controlled)
[39]
[40]
Available in the course of 2004
Current Opinion in Pharmacology 2004, 4:403–407
406 Immunomodulation
immunodeficient syngeneic recipients (NOD SCID mice) by diabetogenic lymphocytes (i.e. from the spleen of overtly diabetic NOD mice) [17,33,34]. The proportions of CD4þCD25þ T cells in CD3-specific antibodytreated tolerant mice were increased in pancreatic and mesenteric lymph nodes, but not in the spleen [33]. More recent data point to the heterogeneity of these CD3-specific antibody-induced regulatory T cells. Indeed, they appear to include not only T cells similar to CD4þCD25þ regulatory T cells controlling physiological autoreactivity and deriving from thymic CD4þCD25þ precursors [35], but also T cells deriving from peripheral CD4þCD25-precursors [33]. Thus, CD3-specific antibody treatment also induces diabetes remission in NOD mice that are invalidated for the costimulation molecule CD28 (NOD CD28/) and are devoid of the thymic natural suppressor CD4þCD25þ population [33]. Another important observation is that the immunoregulatory cytokine transforming growth factor (TGF)-b appears to be a key player in this T cellmediated regulation, although its precise role (i.e. as a mediator of regulation or a growth and/or differentiation factor for regulatory T cells) must still be determined. Thus, CD4þ T cells from CD3-specific-treated tolerant mice consistently produce high levels of TGF-b, and in vivo neutralisation of TGF-b upon injection of specific monoclonal antibodies fully prevents CD3-specific antibody-induced remission [33]. Clinical trials are underway in the attempt to search for adequate modalities that will reproduce this remarkable effect in individual patients. Table 1 provides the details of these studies. Phase I trials were first carried out with the two antibodies mentioned above, OKT3g1 Ala-Ala and ChAglyCD3, in renal allograft recipients presenting acute rejection episodes, and results confirmed that their administration did not elicit major side effects [36,37]. A favourable trend in terms of reversal of the rejection episodes was also observed. On the basis of data obtained in the NOD mouse model, trials are also being conducted on patients presenting recent-onset autoimmune insulindependent (or type I) diabetes [38]. Results from an open trial using the OKT3g1 Ala-Ala antibody provided very encouraging results. One year after short-term treatment, a significant preservation of the b cell mass was observed in treated patients compared with controls [38]. The results of a European multicentric randomized placebo-controlled trial using the ChAglyCD3 antibody in autoimmune diabetes, which we are conducting in collaboration with diabetology centers in Belgium (Belgium Diabetes Registry; Clinical coordinator, B Keymuelen) and Germany (A Ziegler, Munich), will be available over the course of 2004. Interesting data have also been reported recently using OKT3g1 Ala-Ala in psoriatic arthritis [39] and in recipients of islet allografts [40]. Current Opinion in Pharmacology 2004, 4:403–407
Conclusions The introduction of CD3-specific monoclonal antibodies in 1981 in clinical transplantation was a major breakthrough in the field of immunosuppression. For the first time, it was possible to selectively target a T cell surface receptor playing a major functional role. Almost 25 years later, humanised non-mitogenic CD3 antibodies are amongst the most promising tools being used to approach the complex problem of inducing immunological tolerance in the clinic. The capacity of CD3-specific antibodies to trigger TGFb-dependent regulatory T cells, mediating long-standing active suppression, appears as a central mechanism explaining their unique tolerogenic properties. One might hope that a more refined dissection of the molecular mechanisms underlying this effect will lead to the ability to better adapt the use of CD3-specific antibodies to distinct clinical settings. In addition, gaining further insights into specific signalling pathways will pave the way towards the identification of small molecules sharing identical tolerogenic properties.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256:495-497.
2.
Kung P, Goldstein G, Reinherz EL, Schlossman SF: Monoclonal antibodies defining distinctive human T cell surface antigens. Science 1979, 206:347-349.
3.
Cosimi AB, Colvin RB, Burton RC, Rubin RH, Goldstein G, Kung PC, Hansen WP, Delmonico FL, Russell PS: Use of monoclonal antibodies to T-cell subsets for immunologic monitoring and treatment in recipients of renal allografts. N Engl J Med 1981, 305:308-314.
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Ortho Multicenter Transplant Study Group: A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. N Engl J Med 1985, 313:337-342.
5.
Vigeral P, Chkoff N, Chatenoud L, Campos H, Lacombe M, Droz D, Goldstein G, Bach JF, Kreis H: Prophylactic use of OKT3 monoclonal antibody in cadaver kidney recipients. Utilization of OKT3 as the sole immunosuppressive agent. Transplantation 1986, 41:730-733.
6.
Davis MM, Chien YH: T cell antigen receptors. In Fundamental Immunology. Edited by Paul W. New York: Raven Press; 1999:341-366.
7.
Van Wauwe JP, De Mey JR, Goossens JG: OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J Immunol 1980, 124:2708-2713.
8.
Chatenoud L, Legendre C, Ferran C, Bach JF, Kreis H: Corticosteroid inhibition of the OKT3-induced cytokine-related syndrome–dosage and kinetics prerequisites. Transplantation 1991, 51:334-338.
9.
Abramowicz D, Schandene L, Goldman M, Crusiaux A, Vereerstraeten P, De Pauw L, Wybran J, Kinnaert P, Dupont E, Toussaint C: Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation 1989, 47:606-608. www.sciencedirect.com
Anti-CD3 antibodies: towards clinical antigen-specific immunomodulation Chatenoud 407
10. Hirsch R, Gress RE, Pluznik DH, Eckhaus M, Bluestone JA: Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells. J Immunol 1989, 142:737-743. 11. Ferran C, Sheehan K, Dy M, Schreiber R, Merite S, Landais P, Noel LH, Grau G, Bluestone J, Bach JF et al.: Cytokine-related syndrome following injection of anti-CD3 monoclonal antibody: further evidence for transient in vivo T cell activation. Eur J Immunol 1990, 20:509-515. 12. Alegre M, Vandenabeele P, Flamand V, Moser M, Leo O, Abramowicz D, Urbain J, Fiers W, Goldman M: Hypothermia and hypoglycemia induced by anti-CD3 monoclonal antibody in mice: role of tumor necrosis factor. Eur J Immunol 1990, 20:707-710. 13. Van Lier RA, Boot JH, De Groot ER, Aarden LA: Induction of T cell proliferation with anti-CD3 switch-variant monoclonal antibodies: effects of heavy chain isotype in monocytedependent systems. Eur J Immunol 1987, 17:1599-1604. 14. Hirsch R, Bluestone JA, De Nenno 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, 49:1117-1123. 15. Parlevliet KJ, Ten Berge IJ, Yong SL, Surachno J, Wilmink JM, Schellekens PT: In vivo effects of IgA and IgG2a anti-CD3 isotype switch variants. J Clin Invest 1994, 93:2519-2525. 16. Chatenoud L, Primo J, Bach JF: CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice. J Immunol 1997, 158:2947-2954. 17. Chatenoud L, Thervet E, Primo J, Bach JF: Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc Natl Acad Sci USA 1994, 91:123-127.
anti-T cell OKT3 monoclonal antibody. Eur J Immunol 1982, 12:979-982. 27. Chatenoud L, Baudrihaye MF, Chkoff N, Kreis H, Goldstein G, Bach JF: Restriction of the human in vivo immune response against the mouse monoclonal antibody OKT3. J Immunol 1986, 137:830-838. 28. Wong JT, Colvin RB: Selective reduction and proliferation of the CD4R and CD8R T cell subsets with bispecific monoclonal antibodies: evidence for inter-T cell-mediated cytolysis. Clin Immunol Immunopathol 1991, 58:236-250. 29. Wesselborg S, Janssen O, Kabelitz D: Induction of activationdriven death (apoptosis) in activated but not resting peripheral blood T cells. J Immunol 1993, 150:4338-4345. 30. Chatenoud L, Bach JF: Antigenic modulation: a major mechanism of antibody action. Immunol Today 1984, 5:20-25. 31. Nicolls MR, Aversa GG, Pearce NW, Spinelli A, Berger MF, Gurley KE, Hall BM: Induction of long-term specific tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody. Transplantation 1993, 55:459-468. 32. Plain KM, Chen J, Merten S, He XY, Hall BM: Induction of specific tolerance to allografts in rats by therapy with non-mitogenic, non-depleting anti-CD3 monoclonal antibody: association with TH2 cytokines not anergy. Transplantation 1999, 67:605-613. 33. Belghith M, Bluestone JA, Barriot S, Megret J, Bach JF, Chatenoud L: TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 2003, 9:1202-1208. This report provides direct evidence for the role of CD4þCD25þ and þ CD4 CD25– regulatory T cells in the tolerogenic effect of CD3-specific antibodies. In addition, it is the first demonstration of the central role of the immunoregulatory cytokine TGF-b in this model. 34. Chatenoud L, Salomon B, Bluestone JA: Suppressor T cells–they’re back and critical for regulation of autoimmunity! Immunol Rev 2001, 182:149-163.
18. Hughes C, Wolos JA, Giannini EH, Hirsch R: Induction of T helper cell hyporesponsiveness in an experimental model of autoimmunity by using nonmitogenic anti-CD3 monoclonal antibody. J Immunol 1994, 153:3319-3325.
35. Sakaguchi S: Regulatory T cells: key controllers of immunologic self-tolerance. Cell 2000, 101:455-458.
19. Herold KC, Bluestone JA, Montag AG, Parihar A, Wiegner A, Gress RE, Hirsch R: Prevention of autoimmune diabetes with nonactivating anti-CD3 monoclonal antibody. Diabetes 1992, 41:385-391.
36. Friend PJ, Hale G, Chatenoud L, Rebello P, Bradley J, Thiru S, Phillips JM, Waldmann H: Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 1999, 68:1632-1637.
20. Johnson BD, Mccabe C, Hanke CA, Truitt RL: Use of anti-CD3 epsilon F(ab0 )2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation. J Immunol 1995, 154:5542-5554.
37. Woodle ES, Xu D, Zivin RA, Auger J, Charette J, O’laughlin R, Peace D, Jollife LK, Haverty T, Bluestone JA et al.: Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma1(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 1999, 68:608-616. These two manuscripts ([36,37]) constituted the first demonstration in the clinical setting that humanised Fc-mutated CD3-specific antibodies, which did not efficiently bind Fc receptors, were safe. The possibility of overcoming the cytokine-related ‘flu-like’ syndrome was a major step in the clinical development of CD3-specific antibodies that allowed their use not only in transplantation but also in autoimmunity.
21. Bolt S, Routledge E, Lloyd I, Chatenoud L, Pope H, Gorman SD, Clark M, Waldmann H: The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties. Eur J Immunol 1993, 23:403-411. 22. Alegre ML, Peterson LJ, Xu D, Sattar HA, Jeyarajah DR, Kowalkowski K, Thistlethwaite JR, Zivin RA, Jolliffe L, Bluestone JA: A non-activating ‘humanized’ anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo. Transplantation 1994, 57:1537-1543. 23. Smith JA, Tang Q, Bluestone JA: Partial TCR signals delivered by FcR-nonbinding anti-CD3 monoclonal antibodies differentially regulate individual Th subsets. J Immunol 1998, 160:4841-4849.
38. Herold KC, Hagopian W, Auger JA, Poumian Ruiz E, Taylor L, Donaldson D, Gitelman SE, Harlan DM, Xu D, Zivin RA et al.: Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 2002, 346:1692-1698. First report suggesting that, as observed in the NOD mouse model, a long-standing beneficial therapeutic effect may be obtained following a short CD3-specific antibody course in patients presenting with overt autoimmunity.
24. Smith JA, Tso JY, Clark MR, Cole MS, Bluestone JA: Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy. J Exp Med 1997, 185:1413-1422.
39. Utset TO, Auger JA, Peace D, Zivin RA, Xu D, Jolliffe L, Alegre ML, Bluestone JA, Clark MR: Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial. J Rheumatol 2002, 29:1907-1913.
25. Chatenoud L: CD3-specific antibody-induced active tolerance: from bench to bedside. Nat Rev Immunol 2003, 3:123-132.
40. Hering BJ, Kandaswamy R, Harmon JV, Ansite JD, Clemmings SM, Sakai T, Paraskevas S, Eckman PM, Sageshima J, Nakano M et al.: Transplantation of cultured islets from two-layer preserved pancreases in type 1 diabetes with anti-CD3 antibody. Am J Transplant 2004, 4:390-401.
26. Chatenoud L, Baudrihaye MF, Kreis H, Goldstein G, Schindler J, Bach JF: Human in vivo antigenic modulation induced by the
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Current Opinion in Pharmacology 2004, 4:403–407
Current Opinion in Pharmacology 2004, 4:403–407 This review comes from a themed issue on Immunomodulation Edited by Marc Feldmann and Peter Taylor Available online 19th June 2004 1471-4892/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2004.03.012 Abbreviations IL interleukin TCR T cell receptor TGF transforming growth factor
tive cytotoxic effectors) [2]. On this sole basis in the early 1980s, OKT3 was the first monoclonal antibody that entered clinical practice in transplantation. This was four years before its molecular target, the e chain of the CD3 molecule linked to the T cell receptor (TCR) for antigen recognition, was discovered. The strong immunosuppressive potency of OKT3 was evident from the first clinical trials [3–5], and explained the rapid expansion of its use and its approval by regulatory authorities worldwide by 1984. It is the aim of this brief review to discuss the salient features that have marked the clinical development of CD3-specific antibodies over the past 25 years and the uniqueness of their therapeutic properties. Interestingly, and at variance to what was initially thought, this goes far beyond the field of non-specific immunosuppression to also encompass that of immune tolerance.
Biological properties of CD3-specific antibodies All CD3-specific monoclonal antibodies that have been used in experimental and clinical settings are specific to the e chain of the CD3-specific complex. The CD3 complex is a set of five distinct non-polymorphic polypeptide chains expressed on the T cell surface in association with the TCR for antigen recognition. The chains of the CD3 complex are termed e, z, g and d. The z chain is present as a disulfide-bonded homodimer (z–z) or heterodimer. This heterodimer associates the z chain to Z (an alternate splicing product of the z gene) in mouse cells, and to the g chain of the FceRI and the FcgRIII (CD16) receptors in human cells [6]. The intracellular domains of CD3 polypeptides present immunoreceptor tyrosine-based activation motifs (i.e. one for each g, d and e chain, and three for each z chain) that are involved in signal transduction. At the T cell surface, there are at least two ab heterodimers per TCR/CD3 functional complex ([ab]2[g/de]2[zz]4) [6].
Introduction In the mid 1970s, G Kohler and C Milstein reported the technique of producing murine hybridoma-secreting monoclonal antibodies — homogenous sets of immunoglobulins identical in their physicochemical characteristics and specificity — that won the authors the Nobel prize [1]. A few years later, a monoclonal antibody named OKT3 was produced, recognizing a molecule expressed by all mature human T cells. OKT3 proved highly effective at inhibiting lymphocyte functional capacity (i.e. proliferative responses to transplantation antigens [e.g. alloantigens], as well as the generation of alloreacwww.sciencedirect.com
Mitogenicity In contrast to their potent capacity to inhibit in vitro and in vivo antigen-specific immune responses, CD3-specific antibodies are also endowed with potent mitogenic properties owing to their specificity to the TCR-transducing element. In vitro, this mitogenic potential drives T cell proliferation and cytokine production [7]. In vivo, it promotes what was considered the major side effect linked to murine CD3-specific antibody therapy, namely an acute and severe (although self-limited) ‘flu-like’ syndrome comprising high fever, chills, headache and Current Opinion in Pharmacology 2004, 4:403–407
404 Immunomodulation
gastrointestinal symptoms (i.e. vomiting and diarrhea) from the very first hours following initial CD3-specific antibody administration [8,9]. The syndrome is linked to a massive, although transient, systemic release of several cytokines (i.e. tumour necrosis factor, interferong, interleukin [IL]-2, IL-3, IL-6, IL-10, granulocyte–macrophage colony-stimulating factor and IL-4) [8–12]. The mitogenic capacity of CD3-specific antibodies is monocyte dependent, relying upon the capacity of the Fc antibody portion to interact with Fc receptors on monocyte/macrophages, thereby enhancing cross-linking. Thus, in vitro and in vivo, the mitogenic response varies depending upon the isotype of the murine CD3-specific antibody (IgG2a > IgG1 > IgG2b > IgA); CD3-specific F(ab0 )2 fragments, which lack the Fc portion, are nonmitogenic [13–20]. The fact that non-mitogenic CD3specific antibodies fully retained their therapeutic activity constituted the rationale leading to the design of humanised CD3-specific antibodies with engineered non-Fc receptor binding constant portions [21,22]. It is important to emphasize at this point that non-mitogenic CD3specific antibodies are still able to trigger partial signaling both in vitro and in vivo that is essential to mediate their therapeutic activity [16,23,24].
Modulation Unexpectedly, the in vivo administration of CD3-specific antibodies only led to partial depletion of CD3þ cells. In the spleen and lymph nodes of mice treated with Fc receptor binding CD3-specific antibody (145 2C11), approximately 50% of CD3þ cells were eliminated [25]. The pattern is misleading if one only concentrates on peripheral blood, as is the case in patients, because 30– 60 min after the first OKT3 injection, all T cells disappear [26,27]. This effect is mostly caused by the mitogenic and cytokine-releasing effect mentioned above, which favours cell redistribution within the various compartments, trapping by the reticuloendothelial system (opsonisation) and activation of endothelial cells, leading to increased adhesiveness and lymphocyte marginalisation. Interestingly, mechanisms mediating CD3-specific antibody-induced depletion do not only depend upon the functional capacity of the constant Fc antibody portion (i.e. complement-mediated depletion and antibodymediated cell-mediated cytotoxicity), but also upon the antibody’s fine specificity, leading to redirected T cell lysis upon bridging cytotoxic T cells to the target [28] and the induction of apoptosis (i.e. programmed cell death, to which activated cells appear particularly sensitive) [29]. This explains why some degree of CD3þ T cell depletion is also observed with non-Fc binding CD3-specific antibodies (approximately 20–30% in the spleen and lymph nodes) [14,16]. Remnant (non-depleted) T cells undergo antigenic modulation of CD3/TCR. Thus, binding of the CD3-specific antibody to its receptor promotes microaggregation of the Current Opinion in Pharmacology 2004, 4:403–407
complex, capping and subsequent internalisation or shedding [26,30]. These cells are easily detected as they stain CD3–TCR–CD4þ or CD3–TCR–CD8þ [26,27,30]. Antigenic modulation reverses within a few hours (8–12 h) once the antibody is cleared from the environment [26]. CD3-specific antibody-modulated cells are fully unresponsive to antigen-specific or mitogen stimulation [26].
From murine to humanised CD3-specific antibodies The advent of humanised CD3-specific antibodies was an essential milestone in their clinical development. In addition to decreasing the incidence of sensitization, humanisation of CD3-specific antibodies offered the additional advantage of being able to derive non-mitogenic antibodies that were devoid of the deleterious cytokine-releasing activity but still retained their therapeutic potency [21,22]. Two humanised complementarity determining regiongrafted non-mitogenic CD3-specific antibodies are presently being used in clinical trials. OKT3g1 Ala-Ala was characterised by the group of JA Bluestone and is the humanised non-mitogenic version of OKT3 [22]. It presents two mutations in the Fc human g1 region that lead to a 100-fold decrease in the binding of human Fc receptors when compared with OKT3. It is not mitogenic in vitro and does not promote a significant cytokine release in severe combined immunodeficient mice reconstituted with human splenocytes (hu-SPL-SCID mice) [22]. The second antibody, ChAglyCD3, is derived from the rat YTH 12.5 antibody and was characterised by the group of H Waldmann [21]. It expresses a g1 constant region lacking the CH2 domain glycosylation site. As previously shown for other aglycosylated antibodies, ChAglyCD3 is unable to bind Fc receptors, to activate complement, or to induce T cell proliferation in the presence of human serum [21]. In addition, when injected into mice expressing the human CD3-e chain on T cells as a transgene, ChAglyCD3 induced a significantly reduced release of tumour necrosis factor when compared to the parental antibody [21].
From immunosuppression to tolerance induction OKT3 was first used in clinical practice as an immunosuppressant in association with other conventional chemicals such as corticosteroids, azathioprine and, later, cyclosporin A. As such, it proved extremely potent in both preventing and reversing rejection of various organ allografts, and has been widely used, as is illustrated by over 3000 manuscripts published on the topic. By the mid 1990s, regimens using antibodies devoid of the acute side effects described above became available (i.e. CD25 monoclonal antibodies), which explains why OKT3 was progressively abandoned. www.sciencedirect.com
Anti-CD3 antibodies: towards clinical antigen-specific immunomodulation Chatenoud 405
The two main factors explaining the new wave of interest in the use of CD3-specific antibodies concern both the availability of humanised non-mitogenic CD3-specific monoclonals and the compelling experimental evidence arguing for their unique capacity to promote, in both transplantation and autoimmunity settings, a state of antigen-specific unresponsiveness (i.e. immune tolerance). Experiments performed in a rat transplant model by Hall and colleagues [31,32] first showed that short treatment with a non-mitogenic CD3-specific antibody induced permanent engraftment of fully mismatched vascularized heart grafts. In these rats, as proof of the alloantigenspecific tolerance elicited, secondary donor-matched skin grafts survived indefinitely, compared with third party skin allografts that were normally rejected. In the early post-transplant period, CD3-specific antibody-treated recipients showed a typical Th2 immune deviation as reflected by increased IL-4 and IL-5 mRNA levels in the spleen [32]. Spontaneous autoimmune insulin-dependent diabetes (mediated by autoreactive CD4þ and CD8þ T cells) is observed in non-obese diabetic NOD mice. Ten years ago, our group reported the first data showing that a short five-day low-dose treatment of CD3-specific antibodies could restore self-tolerance to b cell antigens in NOD mice at the time of established disease, thus
inducing long-lasting disease remission without need for insulin treatment [16,17,25]. The effect was specific to b cell autoantigens. Thus, syngeneic islet grafts (normally destroyed within a few days in untreated overtly diabetic NOD females owing to recurrence of the autoimmune attack) survived long-term in mice showing CD3-specific antibody-induced remission that were, however, fully capable of normally rejecting skin allografts [17]. The data collected indicate that the immune mechanisms mediating the tolerogenic capacity of CD3-specific antibodies evolve in two phases: induction and maintenance [25]. The induction phase coincides with CD3-specific antibody administration and is associated with clearing of insulitis and a rapid return to normoglycemia. A transient Th2 polarisation is also observed during this first phase, which appears, however, irrelevant to eliciting the longterm antigen-specific effect, as CD3-specific antibodyinduced long-lasting disease remission was also observed in IL-4 invalidated NOD mice (NOD IL-4/) [25,33]. The maintenance phase is characterised by a state of immunological ‘active’ or ‘dominant’ tolerance involving specialized subsets of regulatory or suppressor T cells. Thus protected mice harbour both diabetogenic T cells and CD4þCD25þ and CD4þCD62Lþ regulatory T cells, as assessed by their capacity to block disease transfer into
Table 1 Clinical use of humanised non-mitogenic CD3-specific antibodies. Clinical indication
Monoclonal antibody
Trial
Number of patients
Results
References
Acute renal allograft rejection
OKT3g1 Ala-Ala treatment for 12 consecutive days (in association with conventional immunosuppressants) ChAglyCD3 treatment for eight consecutive days (in association with conventional immunosuppressants) OKT3g1 Ala-Ala treatment for 14 consecutive days
Phase I
Seven patients on antibody treatment
Good safety profile Reversal of rejection in five out of seven patients
[37]
Phase I
Nine patients on antibody treatment
Good safety profile Reversal of rejection in seven out of nine patients
[36]
Phase I/II
24 (12 active treatment; 12 untreated controls)
[38]
OKT3g1 Ala-Ala treatment for 12–14 consecutive days OKT3g1 Ala-Ala treatment for 12 consecutive days (in association with conventional immunosuppressants) ChAglyCD3 treatment for six consecutive days
Phase I/II
Seven patients on antibody treatment
Phase I
Six patients on antibody treatment
At one year, preservation of b cell mass in nine out of 12 treated patients versus two out of 12 untreated controls Six out of seven patients had 75% improvement in number of inflamed joints Four out of six patients achieved and maintained insulin independence with normal metabolic control
Acute renal allograft rejection
Autoimmune insulindependent diabetes
Psoriatic arthritis
Prevention of islet allograft rejection
Autoimmune insulindependent diabetes
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Phase II 80 (40 active (double blind, treatment; 40 placebo) placebo controlled)
[39]
[40]
Available in the course of 2004
Current Opinion in Pharmacology 2004, 4:403–407
406 Immunomodulation
immunodeficient syngeneic recipients (NOD SCID mice) by diabetogenic lymphocytes (i.e. from the spleen of overtly diabetic NOD mice) [17,33,34]. The proportions of CD4þCD25þ T cells in CD3-specific antibodytreated tolerant mice were increased in pancreatic and mesenteric lymph nodes, but not in the spleen [33]. More recent data point to the heterogeneity of these CD3-specific antibody-induced regulatory T cells. Indeed, they appear to include not only T cells similar to CD4þCD25þ regulatory T cells controlling physiological autoreactivity and deriving from thymic CD4þCD25þ precursors [35], but also T cells deriving from peripheral CD4þCD25-precursors [33]. Thus, CD3-specific antibody treatment also induces diabetes remission in NOD mice that are invalidated for the costimulation molecule CD28 (NOD CD28/) and are devoid of the thymic natural suppressor CD4þCD25þ population [33]. Another important observation is that the immunoregulatory cytokine transforming growth factor (TGF)-b appears to be a key player in this T cellmediated regulation, although its precise role (i.e. as a mediator of regulation or a growth and/or differentiation factor for regulatory T cells) must still be determined. Thus, CD4þ T cells from CD3-specific-treated tolerant mice consistently produce high levels of TGF-b, and in vivo neutralisation of TGF-b upon injection of specific monoclonal antibodies fully prevents CD3-specific antibody-induced remission [33]. Clinical trials are underway in the attempt to search for adequate modalities that will reproduce this remarkable effect in individual patients. Table 1 provides the details of these studies. Phase I trials were first carried out with the two antibodies mentioned above, OKT3g1 Ala-Ala and ChAglyCD3, in renal allograft recipients presenting acute rejection episodes, and results confirmed that their administration did not elicit major side effects [36,37]. A favourable trend in terms of reversal of the rejection episodes was also observed. On the basis of data obtained in the NOD mouse model, trials are also being conducted on patients presenting recent-onset autoimmune insulindependent (or type I) diabetes [38]. Results from an open trial using the OKT3g1 Ala-Ala antibody provided very encouraging results. One year after short-term treatment, a significant preservation of the b cell mass was observed in treated patients compared with controls [38]. The results of a European multicentric randomized placebo-controlled trial using the ChAglyCD3 antibody in autoimmune diabetes, which we are conducting in collaboration with diabetology centers in Belgium (Belgium Diabetes Registry; Clinical coordinator, B Keymuelen) and Germany (A Ziegler, Munich), will be available over the course of 2004. Interesting data have also been reported recently using OKT3g1 Ala-Ala in psoriatic arthritis [39] and in recipients of islet allografts [40]. Current Opinion in Pharmacology 2004, 4:403–407
Conclusions The introduction of CD3-specific monoclonal antibodies in 1981 in clinical transplantation was a major breakthrough in the field of immunosuppression. For the first time, it was possible to selectively target a T cell surface receptor playing a major functional role. Almost 25 years later, humanised non-mitogenic CD3 antibodies are amongst the most promising tools being used to approach the complex problem of inducing immunological tolerance in the clinic. The capacity of CD3-specific antibodies to trigger TGFb-dependent regulatory T cells, mediating long-standing active suppression, appears as a central mechanism explaining their unique tolerogenic properties. One might hope that a more refined dissection of the molecular mechanisms underlying this effect will lead to the ability to better adapt the use of CD3-specific antibodies to distinct clinical settings. In addition, gaining further insights into specific signalling pathways will pave the way towards the identification of small molecules sharing identical tolerogenic properties.
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anti-T cell OKT3 monoclonal antibody. Eur J Immunol 1982, 12:979-982. 27. Chatenoud L, Baudrihaye MF, Chkoff N, Kreis H, Goldstein G, Bach JF: Restriction of the human in vivo immune response against the mouse monoclonal antibody OKT3. J Immunol 1986, 137:830-838. 28. Wong JT, Colvin RB: Selective reduction and proliferation of the CD4R and CD8R T cell subsets with bispecific monoclonal antibodies: evidence for inter-T cell-mediated cytolysis. Clin Immunol Immunopathol 1991, 58:236-250. 29. Wesselborg S, Janssen O, Kabelitz D: Induction of activationdriven death (apoptosis) in activated but not resting peripheral blood T cells. J Immunol 1993, 150:4338-4345. 30. Chatenoud L, Bach JF: Antigenic modulation: a major mechanism of antibody action. Immunol Today 1984, 5:20-25. 31. Nicolls MR, Aversa GG, Pearce NW, Spinelli A, Berger MF, Gurley KE, Hall BM: Induction of long-term specific tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody. Transplantation 1993, 55:459-468. 32. Plain KM, Chen J, Merten S, He XY, Hall BM: Induction of specific tolerance to allografts in rats by therapy with non-mitogenic, non-depleting anti-CD3 monoclonal antibody: association with TH2 cytokines not anergy. Transplantation 1999, 67:605-613. 33. Belghith M, Bluestone JA, Barriot S, Megret J, Bach JF, Chatenoud L: TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 2003, 9:1202-1208. This report provides direct evidence for the role of CD4þCD25þ and þ CD4 CD25– regulatory T cells in the tolerogenic effect of CD3-specific antibodies. In addition, it is the first demonstration of the central role of the immunoregulatory cytokine TGF-b in this model. 34. Chatenoud L, Salomon B, Bluestone JA: Suppressor T cells–they’re back and critical for regulation of autoimmunity! Immunol Rev 2001, 182:149-163.
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37. Woodle ES, Xu D, Zivin RA, Auger J, Charette J, O’laughlin R, Peace D, Jollife LK, Haverty T, Bluestone JA et al.: Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma1(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 1999, 68:608-616. These two manuscripts ([36,37]) constituted the first demonstration in the clinical setting that humanised Fc-mutated CD3-specific antibodies, which did not efficiently bind Fc receptors, were safe. The possibility of overcoming the cytokine-related ‘flu-like’ syndrome was a major step in the clinical development of CD3-specific antibodies that allowed their use not only in transplantation but also in autoimmunity.
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38. Herold KC, Hagopian W, Auger JA, Poumian Ruiz E, Taylor L, Donaldson D, Gitelman SE, Harlan DM, Xu D, Zivin RA et al.: Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 2002, 346:1692-1698. First report suggesting that, as observed in the NOD mouse model, a long-standing beneficial therapeutic effect may be obtained following a short CD3-specific antibody course in patients presenting with overt autoimmunity.
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39. Utset TO, Auger JA, Peace D, Zivin RA, Xu D, Jolliffe L, Alegre ML, Bluestone JA, Clark MR: Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial. J Rheumatol 2002, 29:1907-1913.
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40. Hering BJ, Kandaswamy R, Harmon JV, Ansite JD, Clemmings SM, Sakai T, Paraskevas S, Eckman PM, Sageshima J, Nakano M et al.: Transplantation of cultured islets from two-layer preserved pancreases in type 1 diabetes with anti-CD3 antibody. Am J Transplant 2004, 4:390-401.
26. Chatenoud L, Baudrihaye MF, Kreis H, Goldstein G, Schindler J, Bach JF: Human in vivo antigenic modulation induced by the
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