Mammary gland-specific secretion of biologically active immunosuppressive agent cytotoxic-T-lymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4Ig) in milk by transgenesis

Abstract

A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival. Tolerance induction is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side effects. The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient's immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical transplantation. To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals. Prior to the generation of transgenic farm animals, we tested our strategy in mice. Using the promoter of the sheep β-lactoglobulin gene, we expressed our CTLA4Ig chimeric gene in the mammary gland of transgenic mice. The yield of CTLA4Ig was fivefold higher in transgenic milk than that from transfected cells. Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation. We showed that the production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that were fed with the transgenic milk. The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is feasible; further studies involving farm animals are warranted.

Introduction

With advances in surgical techniques and peri-operative care, organ transplantation can now be performed with minimal surgical mortality to save lives of patients with end-stage organ failure. However, allograft rejection remains a major obstacle to the success of clinical transplantation. To prevent rejection and prolong survival, patients are inevitably administered lifelong with immunosuppressive drugs that are associated with many toxic side effects, including opportunistic infection and cancer development. Therefore, a major challenge in the field of transplantation is to induce donor-specific tolerance and achieve long-term graft survival without a deleterious immunosuppressive regime.

T cell activation is a key step in the initiation of graft rejection. Full activation of T cells requires two signals: one is antigen-specific and based on an interaction of T cell receptors (TCRs) with an antigen-major histocompatibility complex (MHC) on antigen-presenting cells (APCs), and the second is an antigen-non-specific co-stimulatory signal. The lack of a co-stimulatory signal after engagement of the TCR by antigen could result in partial or failed T cell activation that renders T cells unresponsive to further antigen challenge, a state known as T cell anergy. T cell anergy is of prime importance in the induction of antigen-specific tolerance to prevent graft rejection. The primary co-stimulatory signal is delivered by B7 receptors (CD80 and CD86) on APCs after ligation of CD28 and cytotoxic-T-lymphocyte antigen 4 (CTLA4) on T cells. CTLA4 displays 20-fold higher affinity than CD28 in binding to B7 receptors Linsley et al., 1991, Linsley et al., 1994. Unlike CD28, binding of CTLA4 to CD80 or CD86 delivers an inhibitory signal, down-regulating T cell activation (Sebille et al., 2001).

Soluble CTLA4Ig is a fusion protein consisting of the extracellular domain of CTLA4 on the N-terminus and the constant regions (CH2 and CH3) and hinge of the Fc domain of IgG on the C-terminus (Linsley et al., 1994). The immunoglobulin portion of CTLA4Ig allows an efficient purification on affinity chromatography column, production of a dimeric CTLA4 protein and a long circulating half-life in vivo. CTLA4Ig functions as a competitive inhibitor of CD28/B7 pathway, suppressing primary and secondary T cell-dependent antibody responses to foreign antigen. Blockade of CD28/B7 co-stimulation by CTLA4Ig reduces graft rejection and prolongs graft survival in rat cardiac transplantation Guillot et al., 2000, Hayashi et al., 2000, Turka et al., 1992, in islet-cell xenografts in mice Feng et al., 1999, Lenschow et al., 1992, in rat renal transplantation (Tomasoni et al., 2000), in renal and islet transplantation in monkeys Kirk et al., 1997, Levisetti et al., 1997, and in rat intestine transplantation Echizenya et al., 2001, Kurlberg et al., 2000. In some instances, CTLA4Ig has been shown to induce transplantation tolerance (Pearson et al., 1994). Successful use of CTLA4Ig in the prolongation of graft survival in animal models suggested that CTLA4Ig might also be an important therapeutic agent for use in clinical transplantation. Indeed, clinical trials using CTLA4Ig in the treatment of psoriasis and graft-versus-host disease (GVHD) in allogenic bone marrow transplantation have shown promising results Abrams et al., 1999, Abrams et al., 2000, Guinan et al., 1999.

CTLA4Ig can be delivered to the transplant recipient, graft or model animals by either direct intravenous injection of purified protein or by CTLA4-expressing adenovirus. Adenoviral gene transfer carries its own disadvantages and adverse effects. Adenovirus in the host can induce an immune response and results in inflammatory and toxic reactions in patients. Production of antibodies in the patients or pre-existing antibody neutralise the adenovirus and render the gene delivery unsuccessful (Romano et al., 2000). Intravenous injection of purified CTLA4Ig can be used to achieve therapeutic CTLA4Ig levels in the sera of recipients. Indeed, intravenous infusions of soluble CTLA4Ig to patients with psoriasis (a T cell-mediated skin disease) successfully slow down autoimmune disease progression and improve the patients' condition Abrams et al., 1999, Abrams et al., 2000. Multiple injections of large quantities of purified CTLA4Ig protein are required to achieve sustained systemic therapeutic levels of CTLA4Ig in the recipient. Until now, CTLA4Ig is purified from transfected cells, a method that is inefficient and, therefore, expensive for the large-scale production required to supply sufficient quantities for clinical application. An efficient and cost-effective method is needed to produce enough CTLA4Ig to cope with the future demands in clinical applications.

Pigs are frequently used as models to establish and refine surgical skill and to test the efficacies of immunosuppressive regime in transplantation before clinical application. We sought to produce large amount of pig CTLA4 human Ig fusion protein and test its efficacy in the prolongation of graft survival in pig transplantation models. Simons et al. (1987) first reported the use of transgenic technology to express foreign proteins in mouse milk. Thereafter, lactation-specific promoters have been used to express proteins of pharmaceutical interests in large quantities in the milk of animals Table 1A, Table 1B. However, the production of immunosuppressive protein in milk using transgenesis has not been studied. To investigate the practicality of using the mammary gland as a bioreactor to produce large quantity of exogenous “non-self” CTLA4Ig in the milk of transgenic farm animals and to assess if the CTLA4Ig in milk will cause detrimental immunosuppression in transgenic animals, we have tested our strategy in transgenic mice. In this study, using the promoter of the sheep β-lactoglobulin gene to direct the expression of pig CTLA4 human Ig chimeric gene in the mouse mammary gland, we produced pig CTLA4 human Ig fusion protein in mouse milk. One would anticipate that the immunosuppressive agent such as CTLA4Ig in animal milk may induce detrimental systemic immunosuppression in transgenic animals and/or animals that have been fed with the transgenic milk. Our data confirm that the production of “non-self” CTLA4Ig in animal milk using transgenesis is safe and transgenic animals do not develop any symptoms of immunosuppression. The transgene was transmitted through the germline and CTLA4Ig is produced in the milk of transgenic offspring.