ATG) in Bone Marrow Recipients

Home , Thymocyte

Bone Marrow Transplantation, (1999) 23, 779–781  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt Monitoring anti-thymocyte globulin (ATG) in bone marrow recipients

TH Eiermann, P Lambrecht and AR Zander

Department of Transfusion Medicine and Bone Marrow Transplantation Unit, Department of Internal Medicine, University Hospital Eppendorf, Hamburg, Germany

Summary: Patients and methods

The present study was undertaken to acquire a ration- Clinical protocol ale for clinical dose adjustment of anti-thymocyte globulin (ATG) to improve cost effectiveness and safety of Twelve patients (nine chronic myelogenous leukaemia, one graft-versus-host disease prophylaxis. The concen- acute myeloid leukaemia, one acute lymphoblastic leu- tration of rabbit ATG in the serum of 12 patients was kaemia, one plasmacytoma) were studied, average age 41 measured by ELISA and by the inhibitory effect on phy- (range 21–55 years). The average body weight was Ϯ Ϯ tohaemagglutinin-induced blastogenesis. At 10 mg/ml 79 16 kg (x s.d.; range 52–106). Six patients had ATG, 3H-thymidine incorporation was effectively received marrow from a matched unrelated donor and six blocked. Serial two-fold dilution of ATG showed that from an HLA-identical sibling. Conditioning therapy con- this effect decreased in a concentration-dependent sisted of 1200 cGy total body irradiation and 60 mg of manner and was lost at 10 ng/ml ATG. One hundred cyclophosphamide per kg of body weight per day for 2 .microlitres serum taken at day ؊1to؉22 post trans- days, or busulphan, cyclophosphamide and etoposide plant effected signiﬁcant inhibition of the phytohaemag- ATG-Fresenius (30 mg/kg) was administered intravenously − glutinin-response with 49 ؎ 12% c.p.m. (x ؎ s.d.) on to 12 subsequent patients daily in three doses on days 3 − -day ؉1 post transplant compared to 93 ؎ 13% c.p.m. to 1. Additional GVHD prophylaxis consisted of cyclo ,on day ؊1(P Ͻ 0.001, unpaired one-sided t-test). The sporine, methothrexate, mitromidazole and pentaglobulin 9 rabbit-IgG was maximal at a concentration of an IgM-enriched immunoglobulin preparation. ␮l/ml at day 0. Subsequently, it decreased 187 ؎ 907 with time. While rabbit-IgG was detectable for a long Mitogen assay period (eg 160 ␮g/ml at day ؉22 in patient MD), the effect on the phytohaemagglutinin-response of normal Peripheral blood mononuclear cells (PBMC) from healthy mononuclear cells lasted up to 4 days post transplant. volunteers were isolated from 20 ml heparinized human We conclude that 90 mg/kg body weight ATG-Fresenius peripheral blood by centrifugation on Ficoll–Hypaque given prior to marrow transplant leads to sustained T (Lymphoﬂot; Biotest, Dreieich, Germany) using standard cell immunosuppression post transplant. methods. Quadruplicate cultures of 1 × 105 PBMC were Keywords: graft-versus-host disease; immunotherapy; T plated in 96-well round-bottom microtitre plates (Greiner lymphocytes; transplantation 650180, Frickenhausen, Germany) in 100 ␮l RPMI 1640 supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 100 U/ml penicillin/streptomycin (GibcoBRL, Life Technologies, Eggenstein, Germany). Anti-thymocyte globulin (ATG) preparations have been Sera of patients (100 ␮l) treated with rabbit ATG used successfully in transplantation, aplastic anaemia and (Fresenius, Bad Homburg, Germany) at various time points graft-versus-host disease (GVHD).1–3 ATG-Fresenius is a before or after bone marrow transplantation, serial dilution polyclonal serum raised in rabbits against the Jurkat T cell of ATG (range 10 mg/ml–2.4 ␮g/ml), or media control line.4 As indicated by previous reports, standardisation of were added. The cells were stimulated for 48 h at 37°C in vitro and clinical activity of polyclonal ATG is a with 5 ␮g/ml phytohaemagglutinin (PHA-L; Boehringer complex issue.5–8 Mannheim, Mannheim, Germany). The proliferative The present study was undertaken to acquire a rationale response was measured by adding 1 ␮Ci 3H- for clinical dose adjustment to improve cost effectiveness thymidine/well (5 Ci/mmol; Amersham Life Science, and safety of GVHD prophylaxis. Rabbit ATG con- Buckinghamshire, UK) approximately 12 h before har- centration was measured in the serum by ELISA and by vesting the cells. Thymidine incorporation (c.p.m.) was inhibition of phytohaemagglutinin-induced blastogenesis. determined on a standard scintillation counter (Beckman LS 5000 TD, Fullerton, CA, USA).

ELISA Correspondence: Dr TH Eiermann, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany Rabbit ATG was determined in microtitre plates coated Received 10 July 1998; accepted 28 October 1998 with anti-rabbit IgG antibody (Boehringer Mannheim Anti-thymocyte globulin TH Eiermann et al 780 30000 (58 Ϯ 32% c.p.m.) and day +1 post transplant (49 Ϯ 12% c.p.m. = –3154 log10 ATG + 5571 c.p.m., P Ͻ 0.001). r2 = 0.81 25000 The concentration of rabbit-IgG in the serum (curve), mean Ϯ standard deviation showed a maximum of Ϯ ␮ Ϯ 20000 907 187 l/ml (x s.d.) at day 0. Thereafter, rabbit-IgG decreased steadily. Individual samples taken between days +6to+22 were grouped (Ͼ5). The rabbit-IgG concentration 15000 was at 270 Ϯ 129 ␮l/ml. Rabbit-IgG was detectable for a c.p.m. long period; for example, in patient MD, 160 ␮g/ml rabbit- 10000 IgG was observed at day +22 post-BMT, although an effect on the PHA-response of normal PBMC was not observed 5000 in any case after day +4 post transplant.

0 10–7 10–6 10–5 10–4 10–3 10–2 10–1 110102 Discussion

ATG (mg/ml) We report the persistence of rabbit-IgG in vivo for up to 3 Figure 1 c.p.m. dependency of PHA-stimulated PBMC cultured with weeks after ATG administration. In order to permit func- serially increasing concentrations of rabbit ATG starting at 10 mg/ml. tional quantification of ATG in lymphocyte-depleted recipients of bone marrow grafts, ex vivo inhibition on PHA- induced blastogenesis of normal mononuclear cells was 1 754 319) as capture antibody. Non-specific binding was used. This method showed a good dose-response inhibition blocked by 0.1% bovine serum albumin (BSA) in 100 ␮l over 6 logs ATG concentrations (Figure 1). phosphate-buffered saline (PBS). One hundred microlitre In Figure 2, we present the correlation between the rab- test serum (1:8000 and 1:16 000 in PBS/0.1% BSA) or bit-IgG concentration in the serum and the relative inhi- standards (seven serial dilutions of ATG-Fresenius in bition (% c.p.m.) of PHA-response of mononuclear cells in PBS/0.1% BSA, range 200–3.1 ng/ml) were added to the 12 patients. On days 1 and 2 after ATG administration had wells and incubated for 1 h at 20°C. After washing three been stopped, the concentration of ATG was still T cell times in PBS/0.1% BSA, alkaline phosphatase-conjugated inhibitory. Thereafter, this effect disappeared, while rabbit- anti-rabbit IgG (200 ␮l anti-rabbit-IgG-AP 400 mU/ml; IgG was still detectable. Boehringer Mannheim 1 214 632) was incubated for 1 h at A long mean elimination half-life of 29 days (range 14– 20°C and the reaction was developed after washing (three 45) of rabbit-IgG was reported by Bunn et al.10 Differences times PBS/0.1%BSA) with 4-nitrophenyl phosphate (p- in the elimination of individual anti-thymocyte antibody NPP, Boehringer Mannheim 726 923, 726 915). After specificities were seen in a rhesus monkey kidney allograft 30 min at 20°C, the optical density was measured at model by immunoprecipitation of 125I-labeled monkey 405 nm. PBMC lysates.11 Following 50 mg/kg rabbit-ATG on days 0–4 after transplant, antibodies against CD8, MHC class I and CD28 persisted until day +15, while antibodies against Data analysis and statistical procedures CD4 and CD16 disappeared before day +13. Our data are PHA-response was calculated as the mean of quadruplicates in agreement with these observations. and given as a percentage of the maximal response obtained A functional level of T cell inhibitory antibody speci- in the absence of ATG (% c.p.m.). An unpaired one-sided ficities of rabbit ATG-Fresenius, which comprise mainly t-test was used to determine the significance (P value) of antibodies against CD2, CD5, CD7 and CD45,8 was detect- differences at various time points. able on day 0 and +1 post BMT, persisting in some patients until day +4 (data not shown), but it was absent long before lymphocyte recovery in recipients took place. This begins Results at approximately day +14 post transplant.12 For the given overall dose of ATG-Fresenius (90 mg/kg Anti-thymocyte globulin was tested for its ability to inhibit body weight) the result indicates that in vivo T cell immuno- the blastogenic response of normal mononuclear cells to suppressive concentrations were attained even after the the mitogen PHA. A dose-response decrease of the c.p.m. infusion of unmanipulated marrow. Thus, in vivo Tcell could be demonstrated (Figure 1). At 10 mg/ml ATG, 3H- depletion of the graft must have been accomplished at saturat- thymidine incorporation was effectively blocked. Serial ing concentrations of ATG-Fresenius. It remains to be ascer- two-fold dilution of ATG showed that this effect decreased tained whether a further dose reduction can be achieved with- over a wide range and was lost at an ATG concentration out losing the impact on the graft-versus-host reaction. of 10 ng/ml. A relationship between the amount of ATG Anti-thymocyte globulin was first used in bone marrow and the decrease of c.p.m. could therefore be established. transplantation by Storb et al13 to prevent rejection of second Figure 2 shows the mean effect of 100 ␮l serum taken marrow grafts in patients with aplastic anemia. Later, it was at day −1to+22 post transplant from the 12 subjects on recognized that this regimen not only prevented rejection but the PHA response of normal PBMC. Significant inhibition also resulted in a low incidence of GVHD.14,15 Based on this of the PHA-response (bars) was observed on day 0 initial observation, ATG was included in the conditioning Anti-thymocyte globulin TH Eiermann et al 781 100 1200 patients with aplastic anemia: a prospective randomized trial. New Engl J Med 1983; 308: 113–118. 3 Deeg HJ, Lougham TP, Storb R. Treatment of human acute 80 1000 graft-versus-host disease with antithymocyte globulin and

800 g/ml) cyclosporin with or without methylprednisolone. Transplan- 60 µ tation 1985; 40: 162–166. 600 4 Grosse-Wilde H, Jakubowski HD, Eigler FW, Kuwert EK. In 40 % c.p.m. 400 vitro immunoresponsiveness in recipients of cadaveric renal allografts during ATG therapy. Proc EDTA 1981; 18: 481–

20 rabbit-IgG ( 200 485. 5 Gascon P, Zoumbos C, Scala G et al. Lymphokine abnormali- 0 0 ties in aplastic anemia: implications for the mechanism of –1 0 1 2 3 4 5 >5 action of antithymocyte globulin. Blood 1985; 65: 407–413. Days post BMT 6 Raefsky EL, Gascon P, Gratwohl A et al. Biological and Figure 2 Inhibition (% c.p.m.) of PHA-stimulated PBMC incubated with immunological characterization of ATG and ALG. Blood rabbit-IgG from plasma of 12 BMT recipients who had received 30 mg/kg 1986; 68: 712–719. ATG-Fresenius on days −3to−1 (mean Ϯ s.d.). Dashed line: correspond- 7 Taniguchi Y, Frickhofen N, Raghavachar A et al. Antilym- ing rabbit-IgG concentrations in serum measured by an ELISA technique. phocyte immunoglobulins stimulate peripheral blood lymphocytes to proliferate and release lymphokines. Eur J Haematol 1990; 44: 244–251. 8 Bourdage JS, Hamlin DM. Comparative polyclonal antithy- regimen in our transplantation protocol as prophylaxis for mocyte globulin and antilymphocyte/antilymphoblast globulin GVHD.15 The outcome in matched unrelated donor transplan- anti-CD antigen analysis by flow cytometry. Transplantation tation is at the time of analysis 68% overall survival with 1995; 59: 1194–1200. moderate incidence of GVHD and no increase in relapse rate 9 Zander AR, Zabelnia T, Kroeger N et al. Matched unrelated donor transplant with a fivefold GvH prevention – single (Ref. 9, manuscript submitted). center experience. Exp Hematol 1998; 25: 822 (Abstr.). Although ATG-Fresenius was ineffective in the treat- 10 Bunn D, Lea CK, Bevan DJ et al. The pharmacokinetics of 7 ment of aplastic anaemia its clinical efficacy as prophy- anti-thymocyte globulin (ATG) following intravenous infusion laxis for GVHD has been confirmed recently by others.16,17 in man. Clin Nephrol 1996; 45: 29–32. ATG-Fresenius was chosen because with the Jurkat T cell 11 Rebellato LM, Gross U, Verbanac KM, Thomas JM. A com- line the immunizing antigen is standardized and readily prehensive definition of the major antibody specificities in available. This should facilitate the direct detection of anti- polyclonal rabbit antithymocyte globulin. Transplantation Jurkat antibodies in sera from treated patients. Jurkat cells 1994; 57: 685–694. resemble activated T cell blasts, which constitutively 12 Lamb LS, Gee AP, Henslee-Downey PJ et al. Phenotypic and express IL-2 and the IL-2 receptor. Thus, Jurkat cells may functional reconstitution of peripheral blood lymphoytes following T cell-depleted bone marrow transplantation from par- be an appropiate immunogen to elicit antibodies against tially mismatched related donors. Bone Marrow Transplant activated T cells for prevention of GVHD. 1998; 21: 461–471. In conclusion, we show by ex vivo inhibition of T cell 13 Storb R, Weiden PL, Sullivan KM et al. Second marrow trans- proliferation that ATG administration prior to BMT leads to plants in patients with aplastic anemia rejecting the first graft: an immunosuppression of PHA-responses post transplant, use of a conditioning regimen including cyclophosphamide which might be a mechanism for prevention of GVHD. and antithymocyte globulin. Blood 1987; 70: 116–121. 14 Storb R, Etzoni R, Anasetti C et al. Cyclophosphamide com- bined with antithymocyte globulin in preparation for allo- geneic marrow transplants in patients with aplastic anemia. Acknowledgements Blood 1994; 84: 941–949. 15 Horstmann M, Stockschla¨der M, Krueger W et al. We thank U Dominikat and C Schwarz for their expert technical Cyclophosphamide/antithymocyte globulin conditioning of assistance. This work was supported by a grant from the Roggen- patients with severe aplastic anemia for marrow transplan- buck Stiftung fu¨r Krebsforschung, Hamburg. tation from HLA-matched siblings: preliminary results. Ann Hematol 1995; 71: 77–81. 16 Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to con- References ventional bone marrow transplantation with lethal cytoreduc- tion for the treatment of malignant and nonmalignant hemato- 1 Woodruff MFA, Anderson NA. Effect of lymphocyte logic diseases. Blood 1998; 91: 756–763. depletion by thoracic duct fistula and administration of anti- 17 Finke J, Bertz H, Behringer D et al. In vivo rabbit anti-T lym- lymphocyte serum on the survival of skin homograft in rat. phocyte globulin efficiently avoids severe GVHD after allo- Nature 1963; 200: 702–704. geneic BMT from matched or mismatched unrelated donors. 2 Champlin R, Gale RP. Anti-thymocyte globulin treatment in Blood 1997; 90 (Suppl.): 186 (Abstr.).