High-Dose IV Administration of Rasburicase Suppresses Anti-rasburicase Antibodies, Depletes Rasburicase-Specific Lymphocytes, and Upregulates Treg Cells

Abstract

Therapeutic proteins can be potent agents for treating serious diseases, but in many patients these proteins provoke antibody responses that blunt therapeutic efficacy. Intravenous administration of high doses of some proteins induces immune tolerance, but the mechanisms underlying this effect are poorly understood. As a model to study tolerance induction in mice, we used rasburicase, a commercial recombinant uricase used for the treatment of hyperuricemia. Intraperitoneal (i.p.) injection of rasburicase without or with alum adjuvants induced a clear anti-rasburicase antibody response, but intravenous (i.v.) injection did not. The lack of response to i.v. rasburicase was apparently due to active immune suppression since i.v.-treated mice showed blunted antibody and reduced T cell responses to subsequent i.p. injections of rasburicase. This blunted response was associated with a decrease in rasburicase-specific B cell and T cell responses and an increase in proportion of CD4+ FoxP3+ regulatory T cells (Treg) in the spleen. We examined the number of lymphocytes in peripheral blood after rasburicase i.v. injection. Rasburicase caused a transient reduction in B and T cells, but a robust and sustained depletion of rasburicase-specific B cells. Further experiments showed that rasburicase i.v. injection decreased the number of lymphocytes and was associated with apoptosis of both B cells and activated T cells and that the enhanced percentage of Treg cells was likely mediated by a macrophage-dependent pathway. Thus, our data suggest that apoptosis and depletion of antigen-specific B lymphocytes and upregulation of Treg cells may play important roles in the immune suppression induced by intravenous administration of a therapeutic protein.

INTRODUCTION

An increasing body of data has shown that therapeutic proteins play an important role in the treatment of several severe diseases (1,2). A serious problem in the clinical application of this strategy is that therapeutic proteins are potentially immunogenic (3,4,5). In some patients, the repeated administration of these protein drugs can provoke antibody responses that blunt therapeutic efficacy and/or cause hypersensitivity reactions.

Accumulated evidence has shown that under certain circumstances, intravenous administration of high doses of some therapeutic proteins induces immune tolerance instead of antibody responses (6,7,8). One example is recombinant coagulation factor VIII, which has been widely applied in clinics for treatment of patients with hemophilia A (9,10). About 20% of patients with hemophilia A who received FVIII treatment develop FVIII inhibitors (anti-FVIII antibodies) (8,11). In these patients, an immune tolerance induction approach using intravenous administration of the protein drug at high dose often suppresses FVIII inhibitors and allows patients to respond to the standard replacement therapy with FVIII (12). However, the mechanisms behind this suppressive effect are poorly understood.

In experimental models with other protein antigens, it has been suggested that tolerance induced by intravenous administration of high-dose protein antigen-induced may be related to anergy of specific T cells (13), antigen-induced T cell apoptosis and clonal depletion (10,14), upregulation of the function of Treg cells (15), or modulation of cytokine response profile so as to induce tolerogenic rather than immunogenic dendritic cell subsets (CD11c+CD11b+) (15). Other studies showed that soluble antigen-induced B cell apoptosis is independent of complement C4 (16) and that B cell receptor ligation-induced apoptosis does not require Fas (17). To date, there is very limited evidence about the direct impact of intravenous therapeutic proteins on macrophages and antigen-specific B cells.

As a model to study the mechanisms of immune tolerance induced by intravenous therapeutic proteins, we used rasburicase, a commercial recombinant pharmaceutical-grade uricase. Uricase catalyzes the oxidation of uric acid into allantoin, a more water-soluble compound easily excreted by the kidney (18). Uricase is found in nearly all organisms, but not in humans; thus, uric acid is the end product in humans, and its accumulation in the blood leads to gout (18). Rasburicase is a recombinant version of uricase (cloned from a strain of Aspergillus flavus) used for the treatment of acute hyperuricemia of tumor lysis syndrome in patients receiving chemotherapy and has been suggested for the treatment of severe hyperuricemia from other sources (e.g., gout). Rasburicase is a tetrameric protein with identical 34 kDa subunits of 301 amino acids. Of patients who were given rasburicase in various clinical studies, between 11% to 64% developed anti-rasburicase neutralizing antibodies (19), which would impair efficacy in any clinical situation that would require repeat dosing—such as gout. In this report, we induced immune tolerance to rasburicase in mice by intravenous (i.v.) injection of rasburicase and explored the mechanisms underlying this suppression. Our results showed that intravenous administration of high-dose rasburicase induced suppression of anti-rasburicase antibody responses, associated with the depletion of rasburicase-specific B cells and T cells, perhaps involving macrophage-mediated upregulation of regulatory T cells.

MATERIALS AND METHODS

Mice and Cells

Female C57BL/6J mice 8 to 10 weeks old (The Jackson Laboratory) were used for in vivo studies and as donors for primary cells. All mice were used in accordance with the Food and Drug Administration Institutional Animal Care and Use Committee guidelines. All cells were cultured with RPMI 1640 complete medium containing 10% fetal calf serum (FCS).

Reagents and Antibodies

Rasburicase (ELITEK), a commercial therapeutic-grade uricase, was obtained from Sanofi (Bridgewater, NJ). All fluorescent-labeled antibodies for cell surface staining were purchased from BD Bioscience (BD Bioscience, San Jose, CA).

Cell Culture

Spleens harvested from treated mice were mashed in RPMI 1640 media, pressed through a cell strainer, and collected into a 50-ml centrifuge tube. After washing with PBS, the cells were pelleted by centrifugation at 1200 RPM for 8 min, and the pellet was resuspended in 3 ml ACK (ammonium-chloride-potassium) lysing buffer to lyse red blood cells. After a subsequent wash in PBS, the cells were resuspended in RPMI 1640 medium for use.

Induction of Anti-rasburicase Immune Response and Its Suppression

To study the suppression of the anti-rasburicase response, we used a protocol based on one developed for studies of murine experimental autoimmune encephalitis (6,13,15,20,21). Mice were “super-immunized” on day 0 by a single 200 μl intraperitoneal (i.p.) injection of 20 μg of rasburicase containing 1% Alhydrogel® (alum) adjuvant (Cat# vac-alu-250, InvivoGen, San Diego, CA); for induction of i.v. suppression, the mice were treated on days −3, 0, and +3 by tail vein injection with 100 μg rasburicase in 200 μl PBS.

Measurement of Anti-rasburicase Antibodies by ELISA

Anti-rasburicase antibodies in mouse sera were measured by ELISA with reference to a standard curve of high-titer anti-rasburicase antibody prepared as follows: anti-rasburicase sera from rasburicase-super-immunized mice were precipitated by 50% ammonium sulfate. The precipitate was dialyzed against PBS at pH 7.4, dissolved by dilution to 5 mg/ml in PBS, and then loaded onto a Protein A affinity column. After the column was washed with PBS, the bound IgG was eluted with 100 mM glycine-HCl, pH 2.8. The eluted IgG was immediately neutralized using 1 M Tris-HCl (pH 9.0) to a final concentration of 0.1 mg/ml. For the ELISA assay, 96-well microtiter plates were coated with 2 μg/well of rasburicase in 100 μl carbonate buffer (pH 9.6). After blocking with 200 μl blocking buffer (PBS-T containing 2% BSA), 100 μl of samples (either undiluted or diluted 1:1000) or serial dilutions of standard (anti-rasburicase IgG, purified as described above) was added to each well, and the plates were incubated for 2 h at room temperature (RT). After washing three times with wash buffer (1X PBS containing 0.05% Tween 20), 100 μl of HRP-conjugated anti-mouse IgG antibody (Cat # SC-516132, Santa Cruz Biotechnology, Dallas, TX) was added to each well and incubated for 1 h at RT. After washing, 100 μl of 3,3′,5,5′-Tetramethylbiphenyl-4,4′-diamine (TMB) substrate solution (Sigma, Cat # T0440, St. Louis, MO) was added to each well for 15 min, followed by the addition of 100 μl stopping buffer (2 M H2SO4). Plates were read at 450 nm with a Molecular Devices plate reader (Sunnyvale, CA). Analysis of the ELISA results under these conditions demonstrated a lower limit of detection of 0.5 ng/ml and a lower limit of quantification of 2 ng/ml. Standard curves routinely exhibited an R2 value >0.994 based on a four-parameter logistic fit of the data.

Flow Cytometric Analysis

To quantify rasburicase-binding cells, rasburicase was labeled with FITC using a Pierce™ FITC Antibody Labeling Kit (Cat# 53027, ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions, and nominally, 50 μg of the labeled rasburicase in 100 μl was subsequently infused i.v. into either naïve or rasburicase-treated mice. Blood and spleens from infused mice were harvested 3 h post infusion, and spleen cells were isolated as described earlier. To gate for specific cell phenotypes, all cells from spleens or peripheral blood samples were treated with red blood cell lysis buffer (except cells harvested from culture) and then were washed with cold PBS containing 1% bovine serum albumin (BSA) and 0.02% NaN3. Cells were stained with fluorescently labeled antibodies against mouse CD3 (Cat #s 562286 or 533064, BD Biosciences), CD4 (Cat #s 553046 or 561091, BD Biosciences), CD8 (Cat #s 553031 or 553035, BD Biosciences), CD25 (Cat # 553071, BD Biosciences), CD11b (Cat # 557657, BD Biosciences), CD11c (Cat # 562783, BD Biosciences), and CD19 (Cat #s 533785, 561738, or 563148, BD Biosciences) as described previously (22). To detect cell apoptosis, cells were stained with APC-Annexin V and propidium iodide (PI) (Cat # 561012, BD Biosciences) according to the manufacturer’s instructions. Treg cells were stained with the Mouse Regulatory T Cell Staining kit (Cat# 88-8111-40, eBioscience, San Diego, CA) according to the manufacturer’s instructions. The levels of Treg cells were presented as percentage of CD4+ FoxP3+ cells in the CD25+ gated cell population. Flow cytometry data were acquired using either FACSCalibur or LSRFortessa (BD Biosciences). Data were analyzed using FlowJo vX.0.7 Software (Tree Star, Ashland, OR).

T Cell Proliferation Assay

To assess T cell proliferation induced by in vitro culture with rasburicase, spleen cells prepared from naive mice were labeled with CFSE (Thermo Scientific, Ashland, OR) according to the manufacturer’s instructions. The labeled cells were incubated in tissue culture plates in the presence of 20 μg/ml of rasburicase or, as a control, with 5 μg/ml of anti-CD3 Ab (Cat # 550275, BD Biosciences) plus 2 μg/ml of anti-CD28 Ab (Cat # 553295, BD Biosciences). To assess T cell proliferation and function in primed mice, spleen cells from animals that had received rasburicase by i.v. injection or had been immunized with rasburicase plus alum adjuvant (i.p.) and then received rasburicase by i.v. injection were cultured in vitro as described above. On day 4 of incubation, cells were harvested and stained with either anti-CD4-APC or anti-CD8-APC (BD Biosciences) and analyzed by flow cytometry for these T cell markers and for CFSE.

Preparation of Peritoneal Macrophages

Macrophages were prepared by peritoneal lavage using a previously described protocol (23). Peritoneal exudate macrophages were harvested by peritoneal lavage from C57BL/6 mice by i.p. injection with 5 ml of PBS. Three minutes after this injection, the peritoneal fluid was withdrawn by using a 10-ml syringe with 19G needle. The harvested fluid was collected in a 50-ml conical tube and centrifuged at 1000 rpm × 10 min. Cells were washed with PBS and resuspended in RPMI 1640 supplemented with 10% FCS. Our data (not shown) and others (24) have indicated that this preparation procedure yields >95% pure CD11b+CD11c macrophages with only 1.8% CD11c+ DCs.

Cytokine Measurements

Cytokine levels in culture supernatants from stimulated and unstimulated spleen cells were determined by multiplex analysis with the Th1/Th2/Th17 Cytokine Beads Array kit (Cat # 560485, BD Biosciences) according to the manufacturer’s instructions. Cytokine measurements for supernatants of macrophage cultures were performed by ELISA array with Mouse Autoimmune Response Multi-Analyte ELISArray Kit (Cat #: MEM-005A, QIAGEN, Germantown, MD) per manufacturer’s protocol.

Induction of Regulatory T Cells in Culture

CD4+ CD25 T cells were isolated from naïve C57BL/6 mice by two-step isolation with magnetic beads (Miltenyi Biotec Inc., San Diego, CA). Briefly, CD25+ cells and CD19+ cells were removed from spleen cells by cell separation with anti-CD19 magnetic beads and anti-CD25-PE plus anti-PE magnetic beads. These negatively selected cells were further incubated with anti-CD4 magnetic beads followed by column separation (Miltenyi Biotec Inc., San Diego, CA). The purified CD4+ T cells were incubated for 3 days in 24-well plates pre-coated overnight with anti-mouse CD3ε and anti-mouse CD28 antibodies as described previously. IL-2 (200 U/ml, Cat # PMC0021, Thermo Scientific) and either TGF-β1 (5 ng/ml, R & D Systems, Cat # 7666-MB-005, Minneapolis, MN) or a 1:5 dilution of the macrophage cell culture supernatants were included in these 3-day cultures (0.5 ml final volume). Anti-TGF-β1 (Cat # MAB1835, R & D Systems) or its IgG1 isotype control (Cat # MAB002, R & D Systems) was added as additional controls. The cells then were transferred to a fresh 24-well plate (without anti-CD3/anti-CD28 coating) in the presence of 200 U/ml of IL-2 for an additional 2 days of culture. The cells were then harvested, and the percentage of CD4+ FoxP3+ cells within total CD25+ gated cells was determined by flow cytometry using the Mouse Regulatory T Cell Staining Kit (Cat # 88-8111-40, eBioscience) per manufacturer’s instructions.

Statistical Analysis

Significance between groups was examined by unpaired two-tailed Student’s t test as indicated in the figure legends. Results are presented as mean ± SD. The significant levels are shown in the figures as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001), respectively.

RESULTS

Intravenous Administration of Rasburicase Induces Suppression of Anti-rasburicase Antibody Responses

Weekly i.p. injection of mice with rasburicase, a clinical-grade recombinant uricase, induced significant levels of anti-rasburicase antibodies (Fig. 1a). However, when initial attempts to elicit antibodies by i.v. injections failed, we decided to test whether i.v. injection with rasburicase might suppress the antibody response to i.p. rasburicase. We used an experimental design similar to an immune tolerance induction protocol in an EAE model (20,21): mice were given three i.v. injections of different doses of rasburicase on days −3, 0, and +3 and weekly i.p injections of rasburicase beginning on day 0. The results showed that i.v. injection with rasburicase reduced specific antibody responses in a dose-dependent manner (Fig. 1b). To develop a rapid immunization protocol for studying the mechanism of this antibody suppression, we “super-immunized” mice on day 0 with a single i.p. injection of rasburicase plus alum adjuvant. Our data showed that the super-immunization induced a more rapid and much higher anti-rasburicase antibody response (Fig. 1c) compared with mice receiving a lower dose of i.p. rasburicase in the absence of alum (Fig. 1b). However, the levels of rasburicase antibodies measured in sera from mice treated with i.v. rasburicase (20 μg) on days −3, 0, and +3 were still markedly reduced. To address the possibility that the apparent inhibition of anti-rasburicase antibody production might actually result from inhibition of the ELISA assay by the rasburicase administered intravenously to the mice, we performed the following control experiment. Mice were super-immunized with rasburicase/alum by i.p. injection on day 0. On day 12, when the mice had developed high levels of anti-rasburicase antibodies, blood samples were collected from the mice, and the mice were then treated with 100 μg rasburicase by i.v. injection. Blood samples were collected from these mice again on the next day. The levels of anti-rasburicase assayed by ELISA in the samples before and after i.v. injection showed no significant difference (p > 0.05, as determined by a two-tailed Student’s t test).

Fig. 1
figure1

Intravenous injection of rasburicase reduced rasburicase-specific antibody responses. a Peritoneal injection of rasburicase induced production of anti-rasburicase antibodies. Mice were given weekly i.p. injections of rasburicase (10 μg/Kg). Peripheral blood samples were collected before each injection for detection of anti-rasburicase antibodies by ELISA. Pooled results are shown from three independent experiments, each consisting of four or five mice. b Intravenous injection of rasburicase reduced subsequent rasburicase i.p.-induced antibody responses in a dose-dependent manner. Each group of four mice was injected intravenously on days −3, 0, and +3 with a different dose (as shown) of rasburicase. Beginning with day 0, the mice were given weekly i.p. injections of rasburicase (10 μg/Kg). Blood samples were collected before each weekly i.p. injection, and anti-rasburicase antibodies were detected by ELISA. Results of three independent experiments are shown as the mean of serum rasburicase antibodies (μg/ml) in each group. c To explore i.v-induced immune suppression, mice (four per group) were intravenously injected with 100 μg of rasburicase on days −3, 0, and +3, respectively, and were immunized on day 0 with a single i.p. injection of 20 μg rasburicase mixed with an equal volume of 2% alum adjuvant (total 200 μl/mouse). Blood samples were collected on the day indicated, and anti-rasburicase antibodies were determined by ELISA. The results of three independent experiments are shown as the mean ± SD of anti-rasburicase antibody from each group

Intravenous Injection of Rasburicase Reduced the Number of Antigen-Specific B and T Cells

To explore the cellular mechanisms underlying intravenous rasburicase-mediated decrease of anti-rasburicase antibodies, we super-immunized mice on day 0 with or without rasburicase i.v. treatment on days −3, 0, and +3. On day 21 after super-immunization, we harvested the spleens and used flow cytometry to assess rasburicase-specific B cells, in vitro rasburicase-induced T cell proliferation as measured by CFSE, and the percentage of CD4+ FoxP3+ cells in the CD25+ T cell population. The results indicate that i.v. rasburicase treatment significantly decreased the levels of rasburicase-specific B cells (Fig. 2a) and the T cell proliferation induced by culture with rasburicase in vitro (Fig. 2b), presumably reflecting decreased proliferation of rasburicase-specific T cells. Interestingly, the percentage of regulatory T cells was enhanced in i.v. rasburicase-treated mice (Fig. 2c). These data suggest that i.v. rasburicase treatment caused reductions in antigen-specific B and T cells as well as upregulation of Treg cell function; these changes may play important roles in i.v. rasburicase-induced antibody suppression. In other experiments, we found that i.v. rasburicase treatment did not induce significant changes in the number of cells with dendritic cell costimulatory molecules CD40, CD80, CD86, and PD-L1, as determined by flow cytometry (Supplementary Figure S1).

Fig. 2
figure2

IV injection of rasburicase decreased rasburicase-specific B cells and reduced T cell proliferation but enhanced percentage of regulatory T cells in the spleen. Mice were treated with rasburicase by i.v. injection on days −3, 0, and +3 and immunized by i.p. injection of rasburicase in alum adjuvant on day 0 as described in Fig. 1c. Mice were sacrificed on day 21 post i.p. immunization, and spleen cells harvested for analysis. a The levels of rasburicase-specific B cells as a percent of total spleen cells were determined by rasburicase-FITC staining and flow cytometry. The results of three separate experiments each using four animals per group are shown as the mean ± SD of the percentage of FITC+ cells for each treatment group. b To analyze T cell proliferation induced by rasburicase, spleen cells from the treated mice were in vitro labeled with CFSE and stimulated by rasburicase (20 μg/ml) for 4 days in culture. T cells proliferating in the presence of rasburicase were analyzed by flow cytometry. The results of three separate experiments each using four animals per group are shown as the mean ± SD of the percentage of proliferating T cells for each treatment group. c The percentage of splenic regulatory T cells was analyzed by flow cytometry using CD4, CD25, and FoxP3 as markers. The levels of regulatory T cells are shown as the percentage of CD4+ FoxP3+ cells amongst the CD25+ gated cells. The results are presented as described above

Intravenous Injection of Rasburicase Depleted Both B Cells and T Cells from Peripheral Blood, Associated with Apoptosis in Both Populations

To assess whether i.v. rasburicase could induce B or T cell effects in mice with an ongoing vigorous anti-rasburicase immune response, we super-immunized naïve mice with i.p. rasburicase + alum 12 days prior to administering 100 μg i.v. rasburicase (the same dose used in our previous experiments) on days −3, 0, and +3 relative to super-immunization. Peripheral blood was then collected at various times after the i.v. treatment to assess the numbers and apoptotic status of B cells and T cells. This delayed i.v. injection of rasburicase resulted in transient declines in the total number of B cells and T cells at 6 h (Fig. 3a, b, d), though upon recovery, there was a significant rebound over pretreatment values. Significantly, the levels of rasburicase-specific B cells remained extremely low long after the i.v. rasburicase injection (Fig. 3c). Consistent with the observed decrease in B cells and T cells, the percentage of apoptotic cells in these populations transiently increased at 6 h after rasburicase i.v. injection (Fig. 3e, f), suggesting that these apoptotic lymphocytes represent rasburicase-specific B and T cells. In contrast with the increase in Treg cells observed with our protocol of i.v. injections on days −3, 0, and +3 (described earlier, Fig. 2c), no significant change in Treg levels was observed at any of the tested time points after the single delayed i.v. injection of rasburicase.

Fig. 3
figure3

IV rasburicase mediated depletion of circulating B cells and T cells. Mice (four per treatment group) were immunized with rasburicase plus alum adjuvant by i.p. injection. On day 12 post immunization, the mice were intravenously injected with 100 μg rasburicase. Blood samples were subsequently collected at the times indicated. ad Total and specific cell counts in peripheral blood as determined by flow cytometry are presented as cell number/100 μl of blood. Data are shown as the mean ± SD and are based on three independent experiments. e, f Apoptotic B cells and T cells were assessed by co-staining cells with Annexin V and PI and analyzing cells by flow cytometry while gating for B cell (e) or T cell (f) markers. Data are shown as the mean ± SD of percentage of apoptotic cells in each treatment group performed in triplicate

Anti-rasburicase Enhanced Rasburicase-Mediated Apoptosis of B Cells and T Cells in Culture

Given that rasburicase/alum i.p. injection can induce production of anti-rasburicase antibodies (Fig. 1c), we speculated that when rasburicase is subsequently injected intravenously, it may form immune complexes (ICs) with circulating anti-rasburicase antibodies and that these ICs might induce suppression of antibody production. To evaluate the role of anti-rasburicase antibodies and their ICs in inducing apoptosis of B cells and T cells, we harvested spleens from naïve control mice and from mice super-immunized 12 days earlier with i.p. rasburicase + alum. The spleens from rasburicase-super-immunized mice yielded spleen cell suspensions containing about 7.5% rasburicase-specific B cells (Fig. 4a). We treated the spleen cells for 6 h in culture with medium alone, with rasburicase plus anti-rasburicase antibodies (immunoglobulin isolated from high-titer anti-rasburicase mouse sera), or with rasburicase plus an equal amount (in μg/ml) of non-specific mouse immunoglobulin from the sera of naïve mice. Staining with both Annexin V and propidium iodide to identify apoptotic cells revealed that in vitro addition of rasburicase caused an increased percentage of apoptotic B cells in splenic cells from super-immunized mice, which was further enhanced by addition of anti-rasburicase antibodies (Fig. 4b). In contrast, no significant change was observed in apoptosis of B cells from unimmunized mice. T cells from unimmunized mice showed no significant change in apoptotic frequency with any in vitro treatment (Fig. 4c). In the absence of in vitro rasburicase, the percentage of apoptotic T cells was higher in the spleens from immunized mice compared to those from unimmunized mice (Fig. 4c). Although these results appear to be different from those described above (Fig. 3), a direct comparison cannot be made: Fig. 3 represents data derived from peripheral blood cells after i.v. exposure to rasburicase whereas the results presented in Fig. 4 explore the responses of isolated spleen cells exposed to rasburicase during in vitro culture.

Fig. 4
figure4

Rasburicase-induced cell apoptosis in culture. Mice were super-immunized by i.p. administration of rasburicase plus alum on day 0, and spleen cells were harvested on day 12 post treatment. ac Cells were incubated for 6 h in culture with medium alone, with rasburicase plus non-specific Ig (from naïve mice), or with rasburicase plus specific anti-rasburicase Ig. a Specific B cells were identified by FITC-rasburicase staining as described in “Materials and Methods”. b, c Cellular apoptosis was assessed by co-staining cells with Annexin V plus PI and gating by flow cytometry against; b B cells and c T cells. d, e Spleen cells from rasburicase-immunized mice were pre-activated with anti-CD3/anti-CD28 antibodies in vitro and then treated with rasburicase with or without anti-rasburicase antibodies prior to assessing apoptosis; d CD4+ T cells or e CD8+ T cells. The data are presented as the mean percentage of cells ± SD based on three independent experiments performed in triplicate

Antigen-specific T cells generally proliferate in response to cognate antigen stimulation in primary culture (as in a typical T cell proliferation assay) (25,26,27). Yet we observed increased T cell apoptosis after rasburicase treatment both in vivo (by i.v. injection, Fig. 3f) and in vitro in primary cell culture (Fig. 4c), suggesting the possibility that the enhanced apoptosis of T cells we observed might be due to activation-induced cell death (AICD) (28,29). To test this hypothesis, we pre-activated the spleen cells from rasburicase-immunized mice with anti-CD3/anti-CD28 antibodies in vitro (28,29) and then treated the activated cells with rasburicase with or without anti-rasburicase antibodies. Similar to the observation of induced apoptosis in B cells (Fig. 4b), the pre-activated T cells from rasburicase-super-immunized mice also underwent apoptosis in response to the in vitro culture with rasburicase or rasburicase plus anti-rasburicase antibodies (Fig. 4d, e), suggesting that besides antigen, the antigen-antibody complex (or immune complex) plays a role in the antibody suppression induced by i.v. injection of rasburicase.

Macrophages Take Up Intravenously Injected Rasburicase and Its Immune Complexes

Phagocytosis by macrophages has been documented as an important mechanism for the clearance of cellular debris and immune complexes and for subsequent regulation of immune responses (30,31,32). To explore the possible role of phagocytosis of rasburicase in mice with an ongoing immune response to this protein, we intravenously administered FITC-labeled rasburicase to naïve mice and to mice super-immunized with i.p. rasburicase + alum 12 days earlier. Three hours after the i.v. injection, spleens were harvested to localize FITC-rasburicase. In splenic cells from immunized mice, we found that FITC-rasburicase was specifically associated with CD11b+ CD11c cells (Fig. 5a), indicating that i.v.-injected rasburicase was largely co-localized with macrophages (33). In contrast, FITC-rasburicase was not associated with macrophages in non-immunized mice (Fig. 5b) or in OVA-immunized mice (data not shown). These data suggest the existence of some factor in the rasburicase-immunized mice that is required for the localization of FITC-rasburicase to splenic macrophages; this factor could be either anti-rasburicase antibodies or FITC-rasburicase-associated apoptotic cells. Despite consistent localization of the FITC to macrophages in the spleen, no localization was found to macrophages in the lymph nodes (data not shown). In addition, such association between FITC-rasburicase and splenic macrophages was found only when FITC-rasburicase was injected by i.v. but not by i.p. administration, possibly reflecting the different pharmacokinetics commonly observed for proteins administered by these two routes and the related fact that i.p. rasburicase would have to pass through the lymph nodes before reaching the blood stream and then the spleen (34).

Fig. 5
figure5

Rasburicase-positive cells in the spleens of immunized mice. FITC-conjugated rasburicase was infused into either control (naïve) mice (a) or rasburicase-super-immunized mice (b) 12 days after immunization. Spleen cells were harvested at 3 h post infusion, and cells co-stained with fluorescently labeled antibodies against CD3, CD19, CD11b, and CD11c were isolated. The stained cells were assessed by flow cytometry using LSRFortessa. The resulting dot plots are representative of three independent experiments performed in triplicate

Interactions Between Macrophages and Rasburicase–Anti-rasburicase Immune Complexes Modulate the Cytokine Profile of Macrophages

One mechanism by which macrophage uptake of rasburicase could reduce the antibody response to this protein would be via secretion of cytokines that might influence the adaptive immune system. To explore this possibility, we examined whether rasburicase or rasburicase–anti-rasburicase immune complexes could alter cytokine production by macrophages. To obtain sufficient macrophage cells for this experiment, we isolated peritoneal (rather than splenic) macrophages from rasburicase-naïve C57BL/6 mice 4 days after i.p. injection with 2 ml of 3% Brewer thioglycollate medium. The macrophages were then cultured for 24 h with medium alone or with either rasburicase plus Ig control or rasburicase plus anti-rasburicase antibody. Cytokine production from the macrophages was analyzed by cytokine array. We found that macrophages treated with rasburicase plus a non-specific Ig control antibody demonstrated an increase in their secretion of the inflammatory cytokines IL-1β, IL-6, IL-10, and TNF-α (Fig. 6). However, in the supernatants from macrophages treated with rasburicase plus anti-rasburicase—and therefore presumably exposed to the corresponding IC—only increased secretion of the cytokine TGF-β was detected.

Fig. 6
figure6

Cytokine levels in the culture of primed macrophages. Peritoneal macrophages isolated from unimmunized mice were left untreated or cultured with rasburicase plus non-specific Ig or specific anti-rasburicase immunoglobulin. Cell supernatants were collected after 24 h, and cytokines in the supernatants were quantified by cytokine ELISA array. The results are expressed as the fold change over the untreated cells and presented as the mean ± SD of two independent experiments performed in duplicate

Given that TGF-β is a key driver for differentiation of regulatory T cells (35,36,37), we wondered whether the TGF-β in supernatants from IC-treated macrophages might augment the differentiation of Treg cells, thus possibly contributing to antibody suppression by i.v. rasburicase. To test this possibility, we activated naïve CD4+ T cells with plate-bound anti-CD3 + anti-CD28 antibodies and treated these cells with IL-2 plus supernatants from macrophages cultured with either medium alone, rasburicase plus non-specific Ig control antibody, or rasburicase plus anti-rasburicase antibody (IC condition). As shown in Fig. 7, supernatants from cultures with rasburicase plus anti-rasburicase antibody (but not rasburicase plus non-specific control Ig antibody) promoted differentiation of FoxP3+ Treg cells. To confirm that the TGF-β in these supernatants was responsible for the increased differentiation of Treg cells observed, we added neutralizing anti-TGF-β antibody to the supernatants from rasburicase plus anti-rasburicase-treated macrophages. We found that the enhanced differentiation of Treg cells was abolished by the addition of this neutralizing anti-TGF-β antibody (Fig. 8), demonstrating the role of macrophage-secreted TGF-β in the differentiation of the Treg cells.

Fig. 7
figure7

Effects of macrophage supernatants on differentiation of regulatory T cells. Murine CD4+CD25 splenic T cells were isolated by magnetic beads as described in “Materials and Methods”. The isolated cells were stimulated with anti-CD3/anti-CD28 antibodies plus 200 U/ml of IL-2, and separate aliquots were stimulated with either 5 ng/ml recombinant TGF-β or with macrophage supernatants (diluted 1:5). Cells were harvested on day 5 after stimulation, and the percentages of Treg cells were assessed by flow cytometry using the Mouse Regulatory T Cell Staining Kit. Results are presented as dot plots derived from the flow cytometric analysis. A representative of three independent experiments is shown. a Treg cell induction by supernatants from macrophages cultured with medium alone as control, with rasburicase plus non-specific-Ig, or with rasburicase plus anti-rasburicase. b Treg cell induction by recombinant TGF-β (5 ng/ml) versus control

Fig. 8
figure8

Anti-TGF-β antibodies abrogate macrophage supernatant-mediated differentiation of Treg cells. Treg cells were induced and analyzed as described in Fig. 7. To confirm the role of TGF-β in the supernatants of rasburicase/anti-rasburicase-treated macrophages in the induction of Treg cells, either anti-TGF-β antibodies or non-specific-Ig control (15 μg/ml) was added to the culture during induction of Treg cells. Upper panel: T cells were incubated with recombinant TGF-β (5 ng/ml) in the presence of mouse IgG1 isotype control or mouse anti-TGF-β monoclonal IgG1 (15 μg/ml), respectively. A representative of two experiments is shown. Lower panel: Treg cells were incubated with diluted (1:5) supernatants from macrophages treated for 24 h with rasburicase/anti-rasburicase plus 200 U/ml IL-2, in the presence of mouse IgG1 isotype control or mouse anti-TGF-β monoclonal IgG1 (15 μg/ml), respectively. A representative of two experiments is shown

DISCUSSION

Among several approaches for the induction of immune tolerance to protein therapeutics, high-dose intravenous tolerization has achieved experimental and clinical success in several systems—notably for factor VIII therapy—though the mechanisms by which this occurs are not entirely understood (8,38). Our study describes a new model system in which intravenous administration of high-dose rasburicase to mice can significantly suppress the anti-rasburicase antibodies induced by two experimental protocols that normally cause robust anti-rasburicase responses: either repeated weekly i.p. rasburicase or a single i.p. super-immunization of rasburicase plus alum. This suppression resembles immune tolerance in that it is accompanied by a sustained reduction in rasburicase-binding B lymphocytes, but future experiments demonstrating that antibody responses to other antigens are not suppressed by i.v. rasburicase would be necessary to formally verify that our results represent antigen-specific tolerance or at least partial tolerance.

If similar suppression of anti-rasburicase antibodies was demonstrated in humans, this might allow repeated rasburicase administration without provoking inhibitory anti-rasburicase antibodies—a regimen that would be useful in the treatment of gout. For other biotherapeutics, this strategy may not be feasible if high doses of the protein are toxic. In theory, this problem might be overcome by administering a therapeutic that was altered—either chemically or by genetic manipulation—to abolish its bioactivity while maintaining its ability to suppress anti-rasburicase antibody production.

Presumably the antibody suppression—or partial tolerance—that we have observed in our experiments with rasburicase does not involve mechanisms unique to that protein but reflects mechanisms that have evolved to regulate the critical decision all B cells face in responding to antigens—including those in immune complexes: whether to activate or suppress programs of B cell proliferation and antibody production (39). Mechanisms tilting B cells towards one or the other direction have been extensively studied in many contexts including normal tolerance to self-antigens or failed tolerance in autoimmune diseases, normal vs failure of immune responses to foreign antigen including vaccines, and strategies for inducing tolerance to tissue transplants and biological therapeutics. In none of these contexts has a complete mechanistic explanation for immune suppression emerged. Instead, many specific intracellular B cell proteins and a wide variety of inter-related variables have been shown to participate in particular cases of immune activation vs suppression. These variables include antigen-antibody affinity (40), the type of antigen-presenting cells (e.g., conventional vs plasmacytoid dendritic cells, M1 vs M2 macrophages), the phenotype of participating T cells (Th1, Th2, Treg), immune complex size and character (e.g., antibody excess vs antigen excess, antibody glycosylation and isotype) (40,41,42,43,44,45), effects of cytokines and other immune signals (DAMPs and PAMPs) acting through various TLR and other receptors, participation of complement components, effects of adjuvants, and the dose, timing, and route of administration of antigen or of exogenous antibody (see reviews in (46,47,48,49)). Here we describe the first experiments with a novel rasburicase system demonstrating suppression of the antibody response to a medically relevant biopharmaceutical. Though we suggest a possible mechanism underlying these events, much work remains to be done to clarify the potential involvement of some of the mechanistic variables noted above.

When high-dose i.v. antigen was administered around the time of i.p. immunization with rasburicase, the resulting suppression of anti-rasburicase antibody was associated with a decrease in the percentage of rasburicase-specific B cells (i.e., B cells binding to FITC-labeled rasburicase) in both spleen and blood samples, as well as a decrease in the numbers of antigen-specific T cells (defined by proliferation during culture of splenic cells with rasburicase). These events were coupled with an increase in the percentage of Treg cells (FoxP3+ CD4+ cells). In addition, in mice with an ongoing immune response 12 days after super-immunization with rasburicase + alum, i.v. infusion of rasburicase was able to induce a striking and sustained reduction in rasburicase-specific B cells, an effect that could contribute to the observed reduction in rasburicase-specific antibody. Previously studied systems of immune tolerance have implicated Treg cells, induction of anergic T cells (13), and induction of B cell and/or T cell apoptosis. It is not clear how intravenous administration of a protein antigen may lead to upregulation of Treg cells, nor is it clear how such a process might lead to antigen-induced apoptosis in our system (although in other systems, induction of apoptosis in autoreactive B cells by certain Treg cells has been documented to depend on PD-L1 (50) or granzyme/perforin (51).

Antibodies in circulation can bind to infused protein antigen to form ICs which are subsequently cleared by phagocytosis (52,53,54,55,56,57), a process that might also contribute to the reduced levels of specific antibodies observed in blood apart from the effect of reduced synthesis due to depletion of antigen-specific B cells. An interesting finding in this study is the interaction between antigen-antibody complexes and macrophages. In order to trace the processing of infused rasburicase in vivo, we explored the potential interaction of rasburicase with several types of immune cells, including dendritic cells, macrophages, T cells, and B cells. We infused FITC-labeled rasburicase into rasburicase-immunized mice displaying high levels of anti-rasburicase antibodies in their sera. We found that FITC-labeled rasburicase was strongly associated with macrophages, but not other cells. Furthermore, this interaction with macrophages only occurred in rasburicase-immunized mice (i.e., anti-rasburicase antibody positive) but not in naïve or OVA-immunized mice. In addition, this association was only observed when rasburicase was injected intravenously but not intraperitoneally. These observations suggest that rasburicase likely forms ICs with anti-rasburicase antibodies and that these ICs bind to macrophages. Though direct evidence is lacking, it is quite plausible that rasburicase/anti-rasburicase ICs interact with macrophages through Fc-FcR interactions.

To further assess the biological consequence of IC binding to macrophages, we isolated peritoneal macrophages from naïve mice, and after culturing them with rasburicase plus non-specific Ig or rasburicase plus anti-rasburicase ICs, we tested culture supernatants for cytokines. We found that in the presence of rasburicase, non-specific Ig—which presumably could not form ICs with rasburicase—caused a surprisingly strong induction of pro-inflammatory cytokines (IL-1β, IL-6, IL-10 TNF-α, MCP-1, and MIP-1β), an effect that may have resulted from high-affinity FcRs engaging antibodies that are mostly non-IC Ig monomers. In contrast, in the presence of ICs (rasburicase plus anti-rasburicase), which may cause relatively stronger engagement of low-affinity FcRs, macrophages produced much less of the pro-inflammatory cytokines but secreted significantly more TGF-β. Given that TGF-β is a Treg-cell-promoting cytokine and that IL-1-β is a key driver of differentiation of naive CD4+ T cells (Th0) to Th17 cells (and a relatively decreased differentiation of Treg cells), the enhanced TGF-β and decreased IL-1-β production observed would be expected to lead to an upregulation of Treg cells. Indeed, we found that cell culture supernatants from macrophages treated with rasburicase plus anti-rasburicase antibody promoted the differentiation of Treg cells, largely due to the presence of TGF-β in these supernatants (as evidenced by the ability of anti-TGF-β mAbs to neutralize this effect).

These data are consistent with the model that intravenously infused rasburicase binds to anti-rasburicase antibodies to form ICs, which in turn interact with macrophages to modify the cytokine profile of these cells, subsequently promoting the development of Treg cells. Such a biological consequence may account for the upregulation of Treg cells and reduced immune responses observed upon long-term intravenous rasburicase treatment. As already mentioned, this ability of ICs to suppress the immune response through their direct interaction with macrophages is consistent with reports by us and others: studies by Schreiber and Unanue have shown that ICs are capable of suppressing IFNγ-induced tumoricidal and bactericidal activity as well as MHC class II expression in macrophages (58,59,60,61). More recent experiments explored the mechanisms of these suppressive effects, demonstrating that ICs interact directly with the CD64 high-affinity Fc receptor on the surface of the macrophage and thereby initiate a cascade of signals culminating in immune suppression (62,63,64,65). Other studies have implicated the CD32 FcgRIIb receptor—which shows low-affinity binding to ICs—as contributing to immune suppression; genetic defects in FcγRIIb are associated with the defective suppression of anti-self-antigen responses in autoimmune disease (66,67).

On the other hand, several laboratories have reported experimental systems in which antibodies acting together with antigen can actually enhance immune responses (68,69). Indeed, in some models, immune complex formation can cause anaphylaxis (70). Interestingly, some of the pathological immune activation effects of ICs may be mediated by FcγRIIA (71,72,73). The precise combination of parameters influencing the immunological effects of ICs (activation vs suppression) may include the specific antigen, route of administration, dosing regimen, host immune status, IC size, and cytokine milieu, among other (as yet undetermined) factors.

Our experiments have demonstrated that the intravenous administration of a high dose of rasburicase can induce systemic suppression of an ongoing anti-rasburicase antibody response, apparently involving the apoptosis and subsequent depletion of antigen-specific B cells and T cells. The long-term antibody suppression seen in these studies may be due to the development of Treg cells, as the interaction between rasburicase/anti-rasburicase immune complexes and macrophages appears to play a key role in driving the differentiation of Treg cells by their induced production of TGF-β and reduced production of IL-1β. An effect of Treg cells on rasburicase-specific B cells would be plausible in the context of literature reports of Treg modulation of B cell function (74).

The present report describes a new example of immune suppression—apparently mediated by antibody-antigen complexes—that reduces the antibody response to a licensed biological therapeutic. Before this approach might be applied clinically, much further work will be required to explore the mechanisms that might be involved.

In the broadest terms, it would be useful to understand how the immune system generates such different responses to the same protein administered intravenously versus intraperitoneally. Our evidence suggests that uptake of intravascular rasburicase by splenic macrophages may initiate an immunosuppressive program of events. Theoretically, extravascularly administered protein antigen—resulting from intraperitoneal (or subcutaneous administration)—could eventually enter the bloodstream and reach the same population of splenic macrophages. Yet our extravascular (intraperitoneal) rasburicase treatment reliably activated antibody responses rather than suppressing them. As noted above, extravascularly administered protein can enter the bloodstream only after percolating through the lymphatic system and the gantlet of immune cells in the lymph nodes, including actively phagocytic and antigen-presenting cells like macrophages and dendritic cells. Typically, only a fraction of extravascularly administered protein reaches the bloodstream and with a delayed time course compared to protein administered by intravascular (intravenous) route (34). After protein molecules from extravascular administration reach the blood, they can of course reach the spleen, which contains its own immune cells. However, the details of cell phenotypes and the dynamic micro-architecture (75,76) of the spleen are likely to be different from those of the lymph nodes, and these differences—along with the later time course and lower plasma concentrations of extravascularly administered antigen—may account for the failure of any observable immunosuppressive effect from i.p. administration of rasburicase despite its eventual arrival in the spleen. In general, protein therapeutics are thought to be less immunogenic when dosed intravenously than extravascularly (usually subcutaneously), but this is not always true (77), and in no case have the precise cellular and biochemical mechanisms responsible for the B cell activation/suppression “decision” been delineated. In the case of immune suppression induced by high-dose antigen, these variables might include the size distribution of any ICs formed, the specific phenotype of Treg cells and/or macrophage subtypes involved, and the need for other critical APC populations, to name a few. Such mechanistic knowledge would be helpful to inform optimization of the dosing regimen for tolerization, particularly to avoid the risk for ICs to exacerbate specific immune responses, which occurs in some experimental systems as mentioned above. The results of such research may eventually be applicable in clinical strategies to reduce antibody responses to rasburicase and, ultimately, to other therapeutic proteins.

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Acknowledgments

We thank Mark Kukuruga and Adovi Akue in the FDA CBER Flow Cytometric Core Facility for their helpful technical assistance.

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Correspondence to Gerald M. Feldman.

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Xu, H., Feldman, G.M. & Max, E.E. High-Dose IV Administration of Rasburicase Suppresses Anti-rasburicase Antibodies, Depletes Rasburicase-Specific Lymphocytes, and Upregulates Treg Cells. AAPS J 22, 80 (2020). https://doi.org/10.1208/s12248-020-00461-0

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KEY WORDS

  • apoptosis
  • immune suppression
  • lymphocyte
  • macrophage
  • therapeutic protein