In vitro depleting antigen - Specific t cells by a promising toxin-antibody conjugate

Abstract. CD137, expressed on activated T cells, is a reliable surrogate marker for antigen specific T cells. In vitro, we examined the possibility for toxin-conjugated anti-CD137 mAb to kill CD137-expressing T cells. Doxorubicin and fluorescence were conjugated to anti-CD137 mAb. We observed that fluorescence-conjugated anti-CD137 was internalized into activated CD4+ T cells and CD8+ T cells within 4 hours after its treatment and these cell lines constitutively expressed CD137. Doxorubicin-conjugated anti-CD137 mAb induced apoptosis in a majority of CD4+ T cells and CD8+ T cells within 48 hours after treatment compared with control antibodies. We further confirm the specificity of toxin-conjugated anti-CD137 mAb showing that only T cells that expressed CD137 underwent apoptosis by staining with toxin-FITC-mAb conjugates. We also found that toxinconjugated anti-CD137 mAb was able to delete allore-active T cells in vitro. Our result indicates that CD137 targeted delivery of toxin is a good strategy to delete antigen-specific T cells.

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JOURNAL OF SCIENCE OF HNUE Natural Sci., 2011, Vol. 56, No. 7, pp. 124-131 IN VITRO DEPLETING ANTIGEN - SPECIFIC T CELLS BY A PROMISING TOXIN-ANTIBODY CONJUGATE Chu Dinh Toi(∗) Hanoi National University of Education Nguyen Van Diep and Chu Dinh Thien Hanoi University of Agriculture (∗)E-mail: chudinhtoi@hnue.com.vn Abstract. CD137, expressed on activated T cells, is a reliable surrogate marker for antigen specific T cells. In vitro, we examined the possibil- ity for toxin-conjugated anti-CD137 mAb to kill CD137-expressing T cells. Doxorubicin and fluorescence were conjugated to anti-CD137 mAb. We observed that fluorescence-conjugated anti-CD137 was internalized into ac- tivated CD4+ T cells and CD8+ T cells within 4 hours after its treatment and these cell lines constitutively expressed CD137. Doxorubicin-conjugated anti-CD137 mAb induced apoptosis in a majority of CD4+ T cells and CD8+ T cells within 48 hours after treatment compared with control anti- bodies. We further confirm the specificity of toxin-conjugated anti-CD137 mAb showing that only T cells that expressed CD137 underwent apoptosis by staining with toxin-FITC-mAb conjugates. We also found that toxin- conjugated anti-CD137 mAb was able to delete allore-active T cells in vitro. Our result indicates that CD137 targeted delivery of toxin is a good strategy to delete antigen-specific T cells. Keywords: Toxin-antibody conjugate, in vitro depleting. 1. Introduction 4-1BB (CD137) is an inducible costimulatory molecule, expressed on activated T cells (CD4+, CD8+, and NKT) [1-4], activated natural killer (NK) cells, dendritic cells (DCs), eosinophils, and mast cells; but Myeloid-Derived Suppressor Cells (MD- SCs ) fail to express surface CD137 [5]. It is a member of the tumor necrosis factor (TNF) receptor super family. Its ligand, 4-1BBL or CD137L, a member of the TNF family, is expressed on activated macrophages, DCs, and B cells etc. Auto-reactive CD4+ T-cells, with the activated form expresses 4-1BB, play important roles in auto-immune diseases by activating macrophages, producing pro- inflammatory cytokines, and helping B-cells to produce auto-antibodies [2, 3, 6]. 124 In vitro depleting antigen - specific T cells by a promising toxin-antibody conjugate Thus, the administration of agonistic anti 4-1BB mono antibodies was found to be effective in eliminating the certain autoimmune diseases, such as experimental autoimmune encephalitis, lupus-like autoimmunity, rheumatoid arthritis and chronic graft-versus-host disease [2, 6-9]. An alternative approach to modulate immune responses in vivo is to specif- ically target activated sub-populations of T cells with appropriate costimulatory signals. In this regard, 4-1BB 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 [10]. We hypothesis that 4-1BB can be used as an attractive target of toxin-conjuga- -ted anti-4-1BB mAb for selective depletion of antigen specific T cells in vitro. 2. Content 2.1. Materials and methods * Materials Mice: C57BL/6 and (C57BL/6 × DBA/2) F1 (BDF1) (H-2b/d) mice, 6 to 8 weeks of age, were purchased from Korea Charles River (Seoul Korea) and Charles River Orient. Antibodies: Anti-mouse CD137 monoclonal antibodies used in our experiments were isolated and purified from ascites by a protein G column (Sigam-Aldrich, St. Louis, MO). The ascites were collected after the administration of hybrid-doma cells (3E1 and 3H3), a kind gift from Dr. Robert Miller, Emory University, of nude mice, and then were purified. An anti-human CD137 monoclonal antibody (4B4, 4785) was isolated and purified from ascites collected from Balb/c mice in the same manner as the mice. Control IgG was purchased for Sigma-Aldrich Korea. * Methods Flow cytometry: The primary immune cells and the cell line (human or mouse) were pre-incubated in a blocking 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). Internalization of anti-CD137 antibodies into cells - Cell lines: CD137 expressing cell lines, CTLL-R8 and CD137 transfected EL- 4, were incubated for 30 minutes with PE-conjugated-anti-CD137 mAb on ice. After that, the cell was washed three times with PBS to remove PE-anti-CD137 antibodies which were not bound to CD137, and cultured in culture medium (RPMI 1640 with 10% FBS and antibiotics) for 30 minutes, 1hr, 2hrs or 4hrs. Samples were taken at 125 Chu Dinh Toi, Nguyen Van Diep and Chu Dinh Thien the indicated time points and attached to a poly-L-lysine-coated slide and fixed with a mounting solution (Flouromuont G). The intracellular location of PE-anti-CD137 antibodies was determined with a fluorescence microscope (Olympus FV500). - Primary murine T cells: Lymphocytes were isolated from spleen and lymph node. The isolated cells were adjusted to 5 × 106 cells/mL and cultured with anti CD3 mAb at the concentration of 0.2 µg/mL for 24 hours. Then a part of the cells were harvested and strained simultaneously with PE-anti CD137 mAb, FITC-anti CD4 mAb or FITC-CD8 mAb, whereby CD137 expression on CD4+ T cells and CD8+ T cells were detected. After detection of CD137, CD4+ T cells and CD8+ T cells were isolated in pure form from the cultured immune cells using MACs method. PE-fluorescence-labelled anti-CD137 antibodies were bound to the isolated CD4+ T cells and CD8+ T cells at 0.4 µg/mL under 4◦C for 30 minutes and washed three times with PBS to remove the PE-anti-CD137 antibodies not bound to CD137, and then suspended in RPMI 1640 with 10% FBS plus antibiotics for 4 hours. After 4 hours of culturing, the cells were harvested and strained with and FITC-anti CD8- mAb at 4◦C for 30 minutes, and washed three times with PBS. After that, the cells were fixed, washed 3 times with PBC and permeability in 0.25% Triton X100. And then, the cells were strained for 1 hour with FITC-fluorescence labelled anti- EEA-1 antibodies. After the straining, the cells were washed three times with PBS and attached to a poly-L-lysine-coated slide and fixed with a mounting solution, and the intracellular location of PE-anti-CD137 antibodies were determined with a fluorescence microscope. Measurement of apoptosis and inhibition of proliferation by anti-CD137 mAb- toxin conjugates Doxorubicin (a kind of anti-tumor agent) was selected as the toxin, and a complex of an anti-CD137 (clone: 3H3, 3E1) and doxorubicin were made. Isolated lymphocytes WT mice and CD137-depleted mice were treated with 0.2 µg/mL of anti-CD3 mAb for 24 hours. After that, the cultured cells were collected and washed twice with PBS, a small amount of cells (1 × 105 cells) were harvested and fluorescence-strained with PE-anti-CD137 mAb and FITC-anti CD8 mAb or FITC-anti CD4 mAb under 4◦C for 30 minutes. After being strained, the cells were washed twice with PBS, and CD137 expression on CD4+ T cells and CD8+ T cells were detected by a flow cytometry (FACS caliber, BD). When CD137 expression was detected, the cultured cells (1 × 106) were treated with various concentrations of anti-CD137 antibody-toxin complex or toxin alone as a control group, and reacted under 4◦C for 30 minutes. After the reaction, the cells were washed three times with PBS to remove any unbound anti-CD137 antibody-doxorubicin complex. Af- ter washing, the cells were suspended on 0.5 mL of cell culture fluid, and additional culture for 48 hours in 48 well-culture-plates. Then, the cells were collected, strained 126 In vitro depleting antigen - specific T cells by a promising toxin-antibody conjugate with annexin V for 20 minutes, and the percentage of annexin V positive cells was analyzed by a flow cytometry. Also, to reveal a direction association between the anti CD137 antibody-doxorubicin complex and cell apoptosis, CD137 was expressed on mouse T cells in the same manner as above, and the cells were treated with FITC-fluorescence labelled anti-CD137 antibodies and doxorubicin and cultured for 24 hours, after that strained with PE-annexin V, followed by analysis of the rela- tionship between the fluorescence location of the anti CD137 antibody-doxorubicin complex and the annexin V by flow cytometry. For proliferation assay with anti-CD137 mAb-doxorubicin conjugates, stimu- lated lymphocytes (4 × 105/well) by anti-CD3 mAb were cultured with anti-CD137 mAb, anti-CD137 mAb-doxorubicin conjugates, and doxorubicin, respectively, at a concentration of 5 µg/mL in a 96 well culture plate for 48 hours. When the incu- bation time reaches 40 hours, each well was treated with 1uCi of thymidine (3H) labelled with radioactive isotope. After 48 hours of culturing, the amounts of isotope in the cultured cells for each experiment group were compared with each other by a micro beta counter. Statistical analysis: Statistical evaluation of differences between experimental groups was performed with Student’s test. 2.2. Results and discussion 2.2.1. Anti-CD137 antibody as a suitable carrier material for delivering toxins into CD137 positive cells Firstly, we determined whether or not anti-CD137 antibodies are effective in delivering a toxin to target cells. For revealing this matter, we investigated the internalization of CD137 and CD137 antibody complex into mouse cell lines, mouse T cells and human T cells. Figure 1. The internalization of CD137 and CD137 antibody complex into mouse CD137 positive cells 127 Chu Dinh Toi, Nguyen Van Diep and Chu Dinh Thien CD137-expressed cell lines (CTLL-R8, CD137 trasnsfected EL-4) and purified CD4+ T cells or CD8+ T cells activated with anti-CD3 mAb for 24 hrs, were incu- bated with PE-conjugated anti-CD137 mAb for 30 minutes on ice. After removal of non-bound antibodies by washing, cells were cultured at 37◦C for 4 hours. Cells were harvested at the indicated time points and strained with a mAb raised against EEA-1, an early endosomal maker, and analyzed for the sub-cellular localization of CD137 and EEA-1 using confocal microscopy. CD137 expressing EL-4, CTLL-R8 cell lines, and activated CD4+, CD8+ T cells derived from the mouse lymph node, spleens were prepared for testing internal- ization of CD137 antibody and CD137 antibody-toxin complex into CD137 positive cells as the method. Figure 1 shows that the PE-anti-CD137 antibodies on the cell surface at incubation 0 are internalized into the cell lines and T cells (CD4+ T cells and CD8+ T cells) after 4 hours of incubation. In order to test if the internalization is induced by endocytosis, the cells are simultaneously strained with an endocyto- sis maker - EEA1 (early endosome antigen-1). As a result, it is observed that the CD137 molecules, the CD137 and the anti-CD137 antibody complex are internalized by endocytosis. From above results, we find that the anti-CD137 antibody used in this in- vestigation is very suitable as a carrier material for delivering toxins into CD137 expressing cells and depleting the cells. 2.2.2. Doxorubicin-conjugated anti-CD137 antibody selectively depleted CD137 positive cells by apoptosis and inhibited cell proliferation in vitro We determined whether the prepared complex normally binds to CD 137 molecules on the cells. To this end, the complex was labelled with FITC fluo- rescence (CD137-doxorubicin-FITC), and the binding strength of the complex to CD137 was compared with that of the anti-CD137 antibody (anti-CD137-FITC) to CD137 molecule. As shown in Figure 2, the measurement of the binding strength of the anti-CD137 antibody confirmed that even when doxorubicin was conjugated to the anti-CD137 antibody, the complex normally bound to CD137 molecules. CD137 positive cells (CD137 expressing cell line and activated spleen immune cells from mice) were cultured and prepared as described in the method. Those cells were either treated with FITC-fluorescence labelled anti-CD137 antibody-doxorubicin complex and cultured for 24 hours, and then strained with PE-annexin V, followed by analysis of the relationship between the fluorescence location of the anti CD137 antibody-doxorubicin complex and the annexin V by flow cytometry. As we ex- pected, the anti CD137 antibody-doxorubicin complex induced the apoptosis in mice CD137 positive cell line. It was demonstrated that apoptosis by this toxin complex was dose-dependent-manner (Figure 3). 128 In vitro depleting antigen - specific T cells by a promising toxin-antibody conjugate Figure 2. The binding strength of anti-CD137 antibody and anti-CD137 antibody-doxorubicin complex to CD137 molecules The binding strength of anti-CD137 antibody and anti-CD137 antibody-doxoru- -bicin to CD137 were described by the percentages of CD137 expressing cells bound to anti-CD137-FITC and anti-CD137-doxorubicin-FITC, respectively after indicated time points Figure 3. The apoptosis effect of anti-CD137 antibody-doxorubicin complex on the CD137 expressing cell line The percentage of Annexin V positive cells was analyzed by a cytometry flow (Figure 3A). The percentage of apoptosis cells at different concentration of anti- CD137 antibody-doxorubicin complex or doxorubicin (Figure 3B). The T cells were killed depending on the concentration of the toxin complex (Figure 4A). On the contrary, the efficacy of such apoptosis was not observed on the immune cells of the 4-1BB KO mice. Our observations suggested that the intracellular delivery of doxorubicin using the anti-CD137 antibody occurred specifically to CD137 molecule and the doxorubicin- conjugated anti-CD137 antibody was effective in selective depletion of CD137 ex- pressing cells. 129 Chu Dinh Toi, Nguyen Van Diep and Chu Dinh Thien Continuously, we wanted to know whether doxorubicin-conjugated anti-CD137 antibody could inhibit proliferation of specific cells or not. Activated immune cells expressing CD137 were cultured on a 96 well culture plate to suitable concentra- tion, and treated with an anti-CD137 antibody, an anti-CD137 antibody-doxorubicin complex, and doxorubicin, respectively. After indicated incubation time, each well was treated with 1uCi of thymidine (3H) labelled with radioactive isotope. The cell proliferation was determined after 48 hours of culturing by a micro beta counter. The anti-CD137 antibody generally binds to CD137 molecules of activated CD8+ and CD4+ T cells and induces the cell proliferation and cell differentiation [2]. In our experiment conditions when treating the activated T cells with only anti-CD137 antibody, this antibody also induced the proliferation of cells. On the contrary, it was noticeable that the doxorubicin-anti-CD137 antibody complex sup- pressed the immune cell proliferation (Figure 4B). We also determined whether the doxorubicin conjugated anti-CD137 antibody actually binds to CD137 positive cells and induce apoptosis. As shown in Figure 4C, the anti-CD137 antibody-doxorubicin complex was clearly present as positive on the annexin V positive cells of CD8+ T cells. Accordingly, our above results suggested that the anti-CD137 antibody-doxoru- -bicin complex did not induce cell proliferation but was active for inducing cell apoptosis, and this complex induced cell apoptosis selectively on CD137 positive cells. Figure 4. The effect of anti-CD137 antibody-doxorubicin complex on the primary CD137 positive cells The rate of apoptosis cells of CD4+ and T CD8+ cells treated with different doses of anti-CD137 antibody-doxorubicin complex or doxorubicin (A, B). The rate of apoptosis CD8+ T cells bound to doxorubicin conjugated anti-CD137 antibody (C). The results are the means plus SE of at least three independent experiments. **, P < 0.01; ***, P < 0.001. 130 In vitro depleting antigen - specific T cells by a promising toxin-antibody conjugate 3. Conclusion From our above results we have some conclusions: - Anti-CD137 mono antibody could be used as a good carrier material for delivering the toxin to CD137 positive cells in vitro. - Anti-CD137 mAb-toxin conjugates selectively depleted the CD137 expressing cells and inhibited the proliferation of these cells in vitro. REFERENCES [1] Kwon, B.S. and S.M. Weissman, 1989. cDNA sequences of two inducible T-cell genes. Proceedings of the National Academy of Sciences of the United States of America, 86(6), pp. 1963-1967. [2] Vinay, D. and B. Kwon, 2006. Immunotherapy Targeting 4-1BB and its Ligand. International Journal of Hematology, 83(1): pp. 23-28. [3] Croft, M., 2009. The role of TNF superfamily members in T-cell function and diseases. Nat Rev Immunol, 9(4), pp. 271-285. 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