Caroline Mary

IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice

The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1beta recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1beta production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.

Inducing CTLA-4–Dependent Immune Regulation by Selective CD28 Blockade Promotes Regulatory T Cells in Organ Transplantation

An improved method of immunosuppression allows better immune function and prolongs the survival of transplanted organs in nonhuman primates. Increasing Tolerance with Less Toxicity According to Chinese legend, 2500 years ago the physician Pien Ch’iao exchanged the hearts of two warriors, one of strong spirit and weak will and the other of weak spirit and strong will, with the aim of achieving balance in their characters. Of course, without modern surgical techniques and the ability to suppress organ rejection by the recipient’s immune system, such transplants would have been impossible. It was not until the discovery of the powerful immunosuppressant drug cyclosporin that organ transplants between unrelated individuals became routinely successful, beginning in the 1980s. Cyclosporin and some newer similar drugs, however, have toxic side effects, and none of them can stop eventual rejection of the organ, so researchers are seeking other options. Now, Vanhove and colleagues test an alternative immunosuppressive strategy. T cells are a major culprit in the immune response directed against transplanted organs, and therefore a number of immunosuppressive drugs target T cells. Cyclosporin, for example, inhibits calcineurin, a protein phosphatase that under normal conditions increases the expression of interleukin 2, which—among other functions—promotes the proliferation of activated T cells. Certain newer approaches to immunosuppression target the process by which T cells become activated. In general, this process is induced by antigen binding to the T cell receptor and reinforced by costimulatory molecules—CD80 and CD86—binding to the T cell CD28 receptor. Additionally, CD80/86 can bind to the CTLA-4 receptor, resulting in T cell inhibition. CTLA-4 is also required for the function of regulatory T cells (Tregs), which suppress aberrant immune responses and are important for inducing tolerance toward transplanted tissue. Several drugs that block CD80/86 have been developed as a nontoxic alternative to calcineurin inhibitors, but in addition to affecting the CD28-mediated pathway of T cell stimulation, these reagents also inhibit the CTLA-4 signals required for Treg function. To avoid inhibiting CTLA-4–dependent regulation of the immune system while suppressing T cell activation, the Vanhove, Blancho, and Pierson labs aimed to block CD28 function directly in experiments on kidney and heart transplantation in baboons and monkeys. Previous work showed that down-regulation of CD28 activity with an anti-CD28 monoclonal antibody inhibited rejection of organ transplants in rodents. For the new nonhuman primate studies, the researchers used a fragment of a human CD28-specific monoclonal antibody fused to a carrier molecule to increase its half-life in vivo. This fusion antibody, sc28AT, recognizes CD28 from monkeys and baboons as well as humans, and it competes with CD80/86 for binding to CD28. sc28AT increased the number of functional Tregs and reduced chronic rejection of the transplanted tissue in the primates when used together with a calcineurin inhibitor. This is good news because it suggests that the dose of such inhibitors (and their toxic side effects) could be reduced in patients when given with a molecule targeting CD28. It remains to be determined whether this approach offers practical advantages over straight CD80/86 blockade when tested in the clinic. Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4–dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4–dependent immune regulation and promotes allograft survival.

Preclinical efficacy and immunological safety of FR104, an antagonist anti-CD28 monovalent Fab' antibody.

Antagonist anti‐CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA‐4 and PDL‐1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti‐CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti‐CD28 Fab′ antibody fragment presenting a long elimination half‐life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti‐CD3 antibodies or when cross‐linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft‐versus‐host disease, which was prevented by administration of FR104 in a CTLA4‐dependent manner. Interestingly, administration of high doses of CTLA4‐Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA‐4, as compared to CD80/CD86 antagonists.

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.

FR104, an Antagonist Anti-CD28 Monovalent Fab' Antibody, Prevents Alloimmunization and Allows Calcineurin Inhibitor Minimization in Nonhuman Primate Renal Allograft

Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA‐4; PD‐L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti‐CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti‐CD28 Fab antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid‐free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios‐negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Treg‐specific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)‐low or CNI‐free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI‐sparing strategy to B7 antagonists for kidney transplant recipients.

Selective Blockade of CD28-Mediated T Cell Costimulation Protects Rhesus Monkeys against Acute Fatal Experimental Autoimmune Encephalomyelitis

Costimulatory and coinhibitory receptor–ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28–CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4–CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab′ Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the β-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

Clinical efficacy of a new CD28-targeting antagonist of T cell co-stimulation in a non-human primate model of collagen-induced arthritis.

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co‐stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen‐induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA‐4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C‐reactive protein (CRP) and interleukin (IL)‐6 and anti‐collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII‐induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.

Antagonist properties of monoclonal antibodies targeting human CD28: Role of valency and the heavy-chain constant domain

Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28-mediated co-stimulatory signals without impeding the co-inhibitory signals dependent on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization. In this study, we evaluated the impact of different formats of a potentially antagonist anti-human CD28 antibody on T cell activation. In particular, we examined the role of valency and of the presence of an Fc domain, two components that might affect receptor multimerization either directly or in the presence of accessory cells expressing Fc receptors. Among monovalent (Fab’, scFv), divalent (Fab’2), monovalent-Fc (Fv-Fc) and divalent-Fc (IgG) formats, only the monovalent formats showed consistent absence of induced CD28 multimerization and absence of associated activation of phosphoinositol-3-kinase, and clear antagonist properties in T cell stimulation assays. In contrast, divalent antibodies showed agonist properties that resulted in cell proliferation and cytokine release in an Fc-independent manner. Conjugation of monovalent antibodies with polyethylene glycol, α-1-antitrypsin or an Fc domain significantly extended their in vivo half-life without modifying their antagonist properties. In conclusion, these data indicate that monovalency is mandatory for maintaining the antagonistic activity of anti-CD28 monoclonal antibodies.

Selective CD28 Antagonist Blunts Memory Immune Responses and Promotes Long-Term Control of Skin Inflammation in Nonhuman Primates.

Novel therapies that specifically target activation and expansion of pathogenic immune cell subsets responsible for autoimmune attacks are needed to confer long-term remission. Pathogenic cells in autoimmunity include memory T lymphocytes that are long-lived and present rapid recall effector functions with reduced activation requirements. Whereas the CD28 costimulation pathway predominantly controls priming of naive T cells and hence generation of adaptive memory cells, the roles of CD28 costimulation on established memory T lymphocytes and the recall of memory responses remain controversial. In contrast to CD80/86 antagonists (CTLA4-Ig), selective CD28 antagonists blunt T cell costimulation while sparing CTLA-4 and PD-L1–dependent coinhibitory signals. Using a new selective CD28 antagonist, we showed that Ag-specific reactivation of human memory T lymphocytes was prevented. Selective CD28 blockade controlled both cellular and humoral memory recall in nonhuman primates and induced long-term Ag-specific unresponsiveness in a memory T cell–mediated inflammatory skin model. No modification of memory T lymphocytes subsets or numbers was observed in the periphery, and importantly no significant reactivation of quiescent viruses was noticed. These findings indicate that pathogenic memory T cell responses are controlled by both CD28 and CTLA-4/PD-L1 cosignals in vivo and that selectively targeting CD28 would help to promote remission of autoimmune diseases and control chronic inflammation.

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.