Application of a toxin-antibody conjugate to treat acute graft versus host disease in mice models

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2011, Vol. 56, No. 7, pp. 132-137 APPLICATION OF A TOXIN-ANTIBODY CONJUGATE TO TREAT ACUTE GRAFT VERSUS HOST DISEASE IN MICE MODELS Chu Dinh Toi(∗) Hanoi National University of Education Chu Dinh Thien Hanoi University of Agriculture Nguyen Thi Thu Ha University of Ulsan, South Korea (∗)E-mail: chudinhtoi@hnue.com.vn Abstract. In previous studies, we found that doxorubicin-conjugated anti- CD137 mAb selectively killed CD137-expressing T cells in vitro. Therefore, in this work we tried to test the ability of this conjugate in vivo. C57lb/6 mice were induced with acute GVHD for further examination. The kinetic of CD137 (CD137) expression was checked on CD4 and CD8 T cells to choose a suitable time of treatment. We found that the T cells in spleen and lymph nodes of aGVHD mice expressed highest CD137 at the 7th day after transplantation. The mice were completely prevented from aGVHD if they were treated by doxorubicin-conjugeted-anti-CD137 antibody at the 7th day after transplantation. This finding encourages applying our toxin-antibody conjugate to treat humans GVHD. Keywords: Toxin-antibody conjugate, in vivo application, GVHD. 1. Introduction Acute graft versus host disease (aGVHD) is caused by donor-derived T cells reacting to recipient alloantigens, involving many T cell subpopulations [1]. Al- loantigen is an antigen existing in alternative forms in a species, thus inducing an immune response when one form is transferred to members of the species who lack it, typical alloantigens are the blood group antigens. Beside donors Th1 (T helpers) cells play a critical role in aGVHD generation, the scientific evidence has shown the role both for the innate and the adaptive immunity in the multifaceted cascade of immunological events that culminates in clinical aGVHD. T regulatory and Th17 cells are T cell subsets which are distinctive from Th1 cells, are likely involved with aGVHD [2]. 132 Application of a toxin-antibody conjugate to treat acute graft versus... There have been several ways to prevent GVHD up to date. Of which, removal of donor T cells in vivo and/or in vitro is the popular and a promising method [3-4]. Solomon et al. [3] depleted donor T cells from peripheral blood stem cell (PBSC) transplant allografts in vivo using an anti-CD25 immuno-toxin in transplantation for treating GVHD patients with a positive results; on the other hand selective depletion of host-reactive T cells (in vivo T-cell depletion) such as removing GVHD-reacting T cells by T cell subset selection, administering the specific donor T cell antibodies, suicide gene insertion or selective allodepletion, and the adoptive transfer of antigen- specific T cells have reached the stage of clinical trials [4]. Kwon et al. [5] found that although there were differences in the pattern of CD137 expression between CD8+ and CD4+ T cells, CD137 is expressed preferentially on both classes of alloreactive T cells that are being activated during alloimmune responses. Auto reactive CD4+ T-cells, with the activated form expresses CD137, plays important roles in autoimmune diseases by activating macrophages, producing proin- -ammatory cytokines, and helping B-cells to produce autoantibodies [6-8]. An ap- proach to modulate immune responses in vivo is to specifically target activated sub-populations of T cells with appropriate costimulatory signals. In this regard, CD137 represents an attractive target. It is expressed on activated, but not resting, T cells, and ligation of this receptor by either its ligand or agonistic antibodies has been shown to provide a potent costimulatory signal [9]. In previous works, we found that the CD137 mAb - doxorubicin conjugate effectively depleted antigen-specific T cells in vitro. Therefore, we hypothesis this conjugate can work well in vivo and prevent aGVHD. 2. Content 2.1. Material and methods * Material Mice: C57BL/6 and (C57BL/6 × DBA/2) F1 (BDF1) (H-2b/d) mice, 6 to 8 weeks old, were purchased from Korea Charles River (Seoul Korea) and Charles River Orient. Mice were maintained in specific pathogen-free (SPF) conditions at facility of Biomedical Research Center, University of Ulsan. All experiments were approved by the institutional animal care committee. Re-agents: Anti-mouse CD137 monoclonal antibody used in our experiments was isolated and purified from ascites by a protein G column (Sigam-Aldrich, St. Louis, MO), the ascites being collected after the administration of hybridoma cells (3E1 and 3H3). Flouromuont G was purchased from Souther Biotech. PEanti CD137 mAb, FITCanti CD4 mAb or FITC-CD8 mAb were purchased from BD Biosciences Pharmingen (San Diego, CA). Ficoll-PaqueTM Plus was purchased from 133 Chu Dinh Toi, Chu Dinh Thien, Nguyen Thi Thu Ha GE Health Biosciences (Uppsala, Sweden). Anti-CD3 mAbs (human clone: OKT3, monkey clone: FN-18) were purchased from U-cytech Bioscience (Netherlands). Doxorubicin was purchased from Peptron (Daejeon, Korea). Induction of aGVHD: Graft-versus-host disease (GVHD) is generally known to be a disease mediated by donor immune cells, i.e., activated T cells. Specifically, for in vivo experiment, first, BDF1 mice were used as recipient mice in order to induce GVHD, and CD57BL/6 mice were used as donor mice. In our experiments, aGVHD model was used. The BDF1 mice were irradiated at 750 rads, bone marrow cells (5 × 106cell/mouse) and splenocytes (2 × 107 cell/mouse) of donor (C57BL/6) mice were injected into the irradiated mice through the tail veins. * Method Cytometry Flow: The primary immune cells were pre-incubated in a block- ing buffer (PBS containing 2.4G2 mAb/0.2% bovine serum albumin/0.1% sodium azide), and then incubated with the relevant mAbs for 30 minutes at 4◦C. Finally, they were washed twice with staining buffer (PBS containing 0.2%BSA/0.1% sodium azide) and analyzed by FACscan (BD Biosciences Pharmingen, MountainView, CA). Statistical analysis: Statistical evaluation of differences between experimental groups was performed with the log rank test for mice survival curves, and with Student’s t test. 2.2. Results and discussion 2.2.1. Doxorubicin-conjugated anti-CD137 antibody could selectively de- plete CD137 positive cells in vivo For confirming the ability of doxorubicin-conjugated anti-CD137 antibody in vitro and answer our question whether this complex works in vivo, we used acute graft-versus-host disease (GVHD) mice model. As shown in Figure 1, CD137 molecules were expressed on T cells by the induction of aGVHD, and it was demonstrated that the levels of expression of CD137 on CD4+ and CD8+ T cells of lymph nodes reached its peak on the 7th and 8th day after aGVHD induction. The level of CD137 molecule on spleen T cells also had the peak on the 7th and 8th day after induction of disease and remained there. This observation indicated that CD137 positive cells could be depleted by anti-CD13- anibody-toxin complex in vivo. In this study, we used the CD137 costimulatory molecule for developing a method of treating and preventing the disease caused by the activation of CD137- ex- pressing cells, in that the intracellular expression of the CD137 molecular is antigen- specific and selective. With the well-known functions of CD137 and its ligand in physiology and pathology conditions [6, 8], the people are more and more inves- 134 Application of a toxin-antibody conjugate to treat acute graft versus... tigating the application of this knowledge in preventing and treating animal and human diseases. Besides, we are continuously finding out the unknown-roles of these molecules in vitro and in vivo. In our experiment, BDF1 mice were used as recipient mice and CD57BL/6 mice were used as donor mice, aGVHD was induced as described in the method. After GVHD induction the mice were sacrificed every other day to harvest spleens, lymph nodes, and immune cells were isolated for collected samples and strained simultaneously with FITC-anti CD4 mAb plus PE-anti CD137 mAb and FITC-anti CD8 mAb plus PE-anti CD137, whereby CD137 expression on CD4+ T cells and CD8+ T cells was detected by flow cytometry. Figure 1. The CD137 expression of CD4+ T cells and CD8+ T cells in acute graft-versus-host disease (aGVHD) mice model The immune cells, isolated from spleens and lymph nodes of aGVHD mice at indicated times after aGVHD induction, were stained with FITC-anti CD4 mAb or FITC-anti CD8 mAb and PE-anti CD137 for checking CD137 expression by cytometry flow. 2.2.2. Doxorubicin-anti-CD137 mAb conjugate effectively treated aGVHD Based on the above result, we treated aGVHD mice by intraperitoneal in- jection of anti-CD137-anibody-docxorubicin complex at 7 days to detect treatment effects. The GVHDmice were divided into 3 groups including untreated, anti-CD137 antibody treated and doxorubicin-anti-CD13-anibody complex treated group. The mouse groups were treated with indicated concentration of individual agent at in- dicated day from GVHD induction, and then mouse survival and bodyweight were 135 Chu Dinh Toi, Chu Dinh Thien, Nguyen Thi Thu Ha checked every 2-3 days. As indices for treatment effects, mouse body weight changes and survival were monitored. As a result, described in Figure 2, it was confirmed that the control groups (untreated group and anti-CD137 antibody treated group) showed significant decrease in bodyweight and survival, whereas the group admin- istered with the doxorubicin-conjugeted-anti-CD137 antibody showed recovery of body weight and higher percentage of survival (Figures 2A and 2B). A. Changes in bodyweight; B. Percentages of survival Figure 2. The doxorubicin-conjugeted-anti-CD137 antibody effectively treated acute GVHD A. Mouse of therapy group; B. Mouse of control group Figure 3. The doxorubicin-conjugeted-anti-CD137 antibody prevented acute GVHD in mouse model These results were more strongly confirmed in Figure 3, the control group (just injected with anti-CD137 mAb) had significant syndromes of GVHD such as diarrhea, hairless, bodyweight loss (Figure 3B), but the Doxorubicin-anti CD137 mAb conjugate treated group had normal health (Figure 3A). From the above results, we concluded that doxorubicin-conjugeted-anti-CD137 antibody could effectively treat acute GVHD, and further more this complex could 136 Application of a toxin-antibody conjugate to treat acute graft versus... selectively deplete CD137 positive cells in vitro as well as in vivo, and thus was useful in treating a specific disease. 3. Conclusion Anti-CD137 mAb-toxin conjugates worked well to prevent aGVHD in mice models. Because this complex selectively depleted the CD137 expressing T cells which caused this disorder in the mice. REFERENCES [1] Jaksch, M. and J. Mattsson, 2005. The Pathophysiology of Acute Graft-Versus- Host Disease. Scandinavian Journal of Immunology, 61(5), pp. 398-409. [2] Engelhardt, B.G., et al., 2010. Regulatory T cell expression of CLA or [al- pha]4[beta]7 and skin or gut acute GVHD outcomes. Bone Marrow Transplant. [3] Solomon, S.R., et al., 2005. Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation. Blood, 106(3), pp. 1123-1129. [4] Barrett, J., 2006. Improving outcome of allogeneic stem cell transplantation by immunomodulation of the early post-transplant environment. Current Opinion in Immunology, 18(5), pp. 592-598. [5] Cho, H.R., et al., 2004. Blockade of CD137 (CD137)/CD137 ligand interactions increases allograft survival. Transplant International, 17(7), pp. 351-361. [6] Vinay, D. and B. Kwon, 2006. Immunotherapy Targeting CD137 and its Ligand. International Journal of Hematology, 83(1), pp. 23-28. [7] Mittler, R., et al., 2004. Anti-CD137 antibodies in the treatment of autoimmune disease and cancer. Immunologic Research, 29(1), pp. 197-208. [8] Croft, M., 2009. The role of TNF superfamily members in T-cell function and diseases. Nat Rev Immunol, 9(4), pp. 271-285. [9] Martin-Orozco, N. and C. Dong, 2007. Inhibitory costimulation and anti-tumor immunity. Seminars in Cancer Biology, 17(4), pp. 288-298. 137