ATGs: how many?? ATG Fresenius: rabbit serum containing polyclonal antibodies against the ALL-T leukemia line Jurkat. TMG Genzyme: rabbit serum containing polyclonal antibodies against human thymocytes.

Antibodies specificities ATG/ALG CD mAb inhibitions Source Fl-labeled CD mAb-50% inhibition point in μg/ml of ATG/ALG CD2 CD3 CD4 CD5 CD7 CD8 ATG AM 130 550 280 1100 150 190 (ATG horse Upjohn) Merieux rabbit 40 230 400 20 70 30 Merieux horse 60 600 400 70 125 130 Fresenius 180 4400 >5000 200 240 1700 MALG 460 1000 1400 500 220 300 (horse ATG Minnesota) Bourdage et al., Transplantation 1995;59:1194-1200

ATG-F / TMG-G: Different spectrum of specifities against co-stimulation molecules activity Activities anti CTLA - 4 ATG - F ++++ TMG - S ++ anti CD 86 ATG - F +++ anti CD 28 TMG - S +++ anti CD 80 ATG - F ++++ TMG - S - Pistillo et al., Transplantation 2000

ATG–F administration schedule (BO) Total dose 15 mg/kg 3 mg/kg CsA - MTX -6 -5 -4 -3 -2 -1 0 Total dose 30 mg/kg 10 mg/kg CsA - MTX -6 -5 -4 -3 -2 -1 0

Prophylaxis of chronic GvHD with or without anti T-lymphocyte globulin (ATG-F) prior allogeneic peripheral stem cell transplantation from HLA-identical sibling after myeloablative conditioning in patients with acute leukemia: A randomised phase III study Primary endpoint: reduction of cGVHD from 60% to 35% Sample size: 60/arm Study medication: ATG-Fresenius 10 mg/kg on days -3-2-1

Protocol Coordinator Germany: Nicolaus Kröger Hamburg EUDRACT-Number 2005-005719-83 (ATGfamily study) Sponsor: University Hospital Hamburg Protocol Coordinator Germany: Nicolaus Kröger Hamburg Protocol Coordinator Italy: Francesca Bonifazi Bologna Scientific Committee: Nicolaus Kröger, Axel R. Zander, Frank Schulz-Kindermann Ernst Holler, Hermann Einsele, Jürgen Finke, Rainer Schwerdtfeger, Michael Schleuning Gerhard Ehninger, Martin Bornhäuser, Hans-Jochem Kolb, Hans-Jochem Kolb Francesca Bonifazi, Giuseppe Bandini, Arnon Nagler, Wolfang Bethke Statistics: Dr. Hinke, Germany Data Management: Andreas Voelp C.R.0. DataLog Clinical monitoring Pharmalog/R. Dautermann 1

Levels of plasma ATG (active) and blood lymphocytes

‘ mean channel value of fluorescence arbitrary units

Half time of Jurkat-reactive antibodies 8 patients after 30 mg/kg ATG-Fresenius days -3, -2, -1.

Preparative regimen ATG – G 800 cGy Tot. dose 7.5 mg 800 Gy From Lowsky et al., 2005 CsA/MMF ATG – G 800 cGy Preparative regimen

ATG-G levels From Lowsky et al., 2005

Intracellular interleukin-4 Comparison of Intracellular Cytokine Production. From Lowsky et al., 2005

Effect of ATG prior to allogeneic stem cell transplantation Before Tx To facilitate engraftment After Tx To reduce risk of GvHD

GVHD after NMA Vs MA AlloSCT: Skin Morbidities involving the skin, liver, and gut after nonmyeloablative conditioning compared with myeloablative conditioning. Liver Gut Figura 2 Overall Acute Chronic Mielcarek et al. Blood 2003

From Sala-Torra et al., 2008

Linfomi.Ric GVHD ACUTA E CRONICA: ATG VS NON ATG (BO) Nr. pazienti

I and II treatment plans ATG-G - 4 - 1 10 mg/KG 200 Gy FK / MTX 3/16 graft failure I - 7 -10 ATG-G 10 mg/KG II no graft failure more infections more relapse 450 Gy FK / MTX - 1 G-csf From Toor et al., 2008

Hematopoietic Stem Cell Transplantation in a canine model cATG-G 2-5 mg/kg 100 Gy Csa /MMF - 12 - 7 From Diaconescu et al., 2005

Dog. N°. cATG-g (mg/kg) Marrow Cells (million/kg) Maximum Donor MNC Chimerism (%) Graft Rejection Engraftment Duration (wk) TNC CD34 CD3 G200 3.5 263 4.2 17.8 5 Yes 8 G198 4 345 4.1 25.1 25 18 G166 206 7.5 6.9 11 G208 453 11.3 38.5 40 No ≥36 G252 175 6.6 13.2 From Diaconescu et al., 2005

Preparative regimen ATG – G 800 cGy Tot. dose 7.5 mg 800 Gy From Lowsky et al., 2005 CsA/MMF ATG – G 800 cGy Preparative regimen

Lymphoid and myeloid engraftment From Lowsky et al., 2005

T-cell engraftment From Lowsky et al., 2005

Conclusioni Utilizzo ATG non sarebbe indicato, in quanto riduce la GVHD, soprattutto cronica, diminuendo la GvL. Utilizzo ATG indicato in quanto necessario per favorire l‘attecchimento, il chimerismo completo, elementi di primaria importanza ai fini del successo (eradicazione della malattia) del trapianto. La tossicità è minima. Gli ATG concorrono a realizzare dei condizionamenti “ridotti” evitando l’impiego di farmaci citotossici/immunosoppressivi. Poiché il punto 2 è molto più importante del punto 1, gli ATG devono essere usati nei regimi “a intensità ridotta”.