RESUMEN
Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.
Asunto(s)
Ácidos Borónicos/uso terapéutico , Proteína Inhibidora del Complemento C1/uso terapéutico , Galactosiltransferasas/genética , Supervivencia de Injerto/fisiología , Xenoinjertos , Trasplante de Riñón , Intercambio Plasmático , Pirazinas/uso terapéutico , Animales , Animales Modificados Genéticamente , Enfermedades Autoinmunes , Bortezomib , Citomegalovirus/fisiología , Galactosiltransferasas/deficiencia , Técnicas de Inactivación de Genes , Inmunidad Innata/fisiología , Inmunosupresores/uso terapéutico , Riñón/cirugía , Riñón/virología , Modelos Animales , Papio anubis , Sus scrofa , Replicación Viral/fisiologíaRESUMEN
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.
Asunto(s)
Antígenos CD28/inmunología , Inhibidores de la Calcineurina/farmacología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Inmunización , Fragmentos Fab de Inmunoglobulinas/farmacología , Trasplante de Riñón , Animales , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Rechazo de Injerto/tratamiento farmacológico , Supervivencia de Injerto/efectos de los fármacos , Técnicas para Inmunoenzimas , Terapia de Inmunosupresión , Enfermedades Renales/inmunología , Enfermedades Renales/cirugía , Papio , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/inmunología , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Antígenos CD28/inmunología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Animales , Antígeno CTLA-4/inmunología , Evaluación Preclínica de Medicamentos , Citometría de Flujo , Fragmentos Fab de Inmunoglobulinas/efectos adversos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunohistoquímica , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Fucosiltransferasas/metabolismo , Trasplante de Riñón/inmunología , Trasplante Heterólogo/inmunología , Animales , Animales Modificados Genéticamente , Retrovirus Endógenos/metabolismo , Rechazo de Injerto , Supervivencia de Injerto , Humanos , Inmunoglobulina G/química , Inmunosupresores/uso terapéutico , Trasplante de Riñón/métodos , Papio , Porcinos , Factores de Tiempo , Trasplante Heterólogo/métodosRESUMEN
Lymphocyte-activation gene-3 (LAG-3, CD223) is a marker for recently activated effector T cells. Activated T lymphocytes are of major importance in many autoimmune diseases and organ transplant rejection. Therefore, specifically depleting LAG-3(+) T cells might lead to targeted immunosuppression that would spare resting T cells while eliminating pathogenic activated T cells. We have shown previously that anti-LAG-3 antibodies sharing depleting as well as modulating activities inhibit heart allograft rejection in rats. Here, we have developed and characterized a cytotoxic LAG-3 chimeric antibody (chimeric A9H12), and evaluated its potential as a selective therapeutic depleting agent in a non-human primate model of delayed-type hypersensitivity (DTH). Chimeric A9H12 showed a high affinity to its antigen and depleted both cytomegalovirus (CMV)-activated CD4(+) and CD8(+) human T lymphocytes in vitro. In vivo, a single intravenous injection at either 1 or 0·1 mg/kg was sufficient to deplete LAG-3(+) -activated T cells in lymph nodes and to prevent the T helper type 1 (Th1)-driven skin inflammation in a tuberculin-induced DTH model in baboons. T lymphocyte and macrophage infiltration into the skin was also reduced. The in vivo effect was long-lasting, as several weeks to months were required after injection to restore a positive reaction after antigen challenge. Our data confirm that LAG-3 is a promising therapeutic target for depleting antibodies that might lead to higher therapeutic indexes compared to traditional immunosuppressive agents in autoimmune diseases and transplantation.