Use of culture to reach metabolically adequate beta

Use of culture to reach metabolically adequate beta cell dose by combining donor islet cell isolates for transplantation in type 1 diabetes patients Da. Hae Lee*, Pieter Gillard*, Robert Hilbrands, Zhidong Ling, Ursule Van de Velde, Daniel Jacobs-Tulleneers-Thevissen, Geert Maleux, Bruno Lapauw, Laurent Crenier, Christophe De Block, Chantal Mathieu , Daniel Pipeleers , Bart Keymeulen BTS, Brussel 2020

Islet transplantation Naftanel MA, Harlan DM (2004) Pancreatic Islet Transplantation. PLo. S Med 1(3): e 58

Transplantation of islet beta-cell grafts FRESH CULTURE Isolation 0 hours 25 +/-10 hrs NEJM, 2002 International, 2012 > 24 -480 hrs PNAS, 2006 Current

Aim Investigate the impact of culture time on longterm graft survival and function in C-peptide negative type 1 diabetic recipients of cultured islet beta-cell grafts

Study Cohort: 40 patients type 1 diabetes mellitus receiving islet transplantation between December 2001 -January 2011 with : • Hypoglycemia unawareness • C-peptide negative • Large variation of blood glucose levels (glycemic variability >25%) • Hb. A 1 c >7% • 1 chronic diabetes complication First Graft Islet cells* combined from 1 -6 donors culture for 16 to 511 hours ATG + TAC +MMF Median 2. 8 months (IQR 2. 2 -3. 2) , 30/40 received second islet infusion Second Graft Islet cells combined from 1 -10 donors cultured for 19 to 821 hours TAC + MMF= 1/30 Main outcome • Duration of plasma c-peptide ≥ 0. 5 ng/ml Secondary outcome • Duration of C-peptide 1 ng/ml in combination with glycemic variability < 25% • Duration of insulin independence

Factors correlated with duration of implant function Parameters Graft characteristics Beta 1 Beta Cell number (x 10 6) Beta cell number /kg body weight (x 10 6) Univariate Estimate p-value Multivariate p-value Estimate 0. 022 0. 891 0. 866 Beta 2 Beta Cell number (x 10 6) Beta Cell number/kg BW (x 10 6) 0. 013 0. 092 0. 994 0. 630 Beta 1 + Beta 2 Beta cell number (x 10 6) Beta cell number/kg BW (x 10 6) -0. 068 -0. 034 0. 679 0. 834 Culture time Beta 1 All cells in culture ≥ 96 hours (yes/no) 0. 486 0. 001 0. 422 0. 005 Patient characteristics Age at transplantation BMI (kg/m²) Hb. A 1 c (%) B-lymphocytes (CD 19) T-lymphocytes (CD 3) Donor-specific HLA antibodies Presence of auto-antibodies (yes/no) T-cell auto-reactivity for GAD (yes/no) T-cell auto-reactivity for IA 2 (yes/no) 0. 313 -0. 273 -0. 292 0. 083 -0. 053 -0. 038 -0. 225 -0. 232 -0. 107 0. 049 0. 088 0. 071 0. 611 0. 733 0. 815 0. 162 0. 166 0. 530 0. 307 0. 036

Study Cohort: 40 patients type 1 diabetes mellitus receiving islet transplantation between December 2001 -January 2011 with : • Hypoglycemia unawareness • C-peptide negative • Large variation of blood glucose levels (glycemic variability >25%) • Hb. A 1 c >7% • 1 chronic diabetes complication First Graft Islet cells* combined from 1 -6 donors culture for 16 to 511 hours All cells cultured at least 96 hours: n= 10/40 Median 2. 8 months (IQR 2. 2 -3. 2) , 30/40 received second islet infusion Second Graft Islet cells combined from 1 -10 donors cultured for 19 to 821 hours All cells cultured at least 96 hours: n= 1/30 Main outcome • Duration of plasma c-peptide ≥ 0. 5 ng/ml Secondary outcome • Duration of C-peptide 1 ng/ml in combination with glycemic variability < 25% • Duration of insulin independence

Baseline characteristics All cells cultured ≥ 96 hours < 100% cells cultured <96 hours First Graft (n=40) 10 30 Body weight (kg) BMI (kg/m 2) Age at transplantation (years) Hb. A 1 c (%) 62. 4 ± 7. 4 22. 0 ± 1. 9 46. 5 ± 6. 4 7. 2 ± 0. 8 70. 3 ± 8. 4 * 24. 3 ± 2. 4 * 45. 4 ± 9. 4 7. 8 ± 0. 9 Total lymphocyte count CD 3+ count CD 19+ count Leucocyte count before ATG CD 4 /CD 8 ratio Auto-antibodies (ICA/GAD/IA 2) Positivity of 2 or more antibodies 1890 ± 996 1384 ± 627 296 ± 155 5993 ± 2764 2. 0 ± 0. 7 0/3/4 4 1822 ± 500 1259 ± 272 236 ± 98 5972 ± 1438 2. 0 ± 1. 0 8/14/7 18 T-cell autoreactivity against: IA 2 and/or GAD (yes) IA 2 (yes) GAD (yes) 4/10 1/10 2/10 19/26 10/26 16/26 Presence of donor-specific HLA antibodies 2 3

Duration of plasma c-peptide ≥ 0. 5 ng/ml

Duration of C-peptide 1 ng/ml + glycemic variability < 25%

Duration of Insulin independence

Cell number and composition of grafts in the first graft First Graft ≧ 96 h in culture < 96 h 10 30 2. 6 (2. 1 -3. 1) 3. 1 (2. 7 -3. 5) 0. 187 Total 491 (392 -591) 757 (665 -850) 0. 001 Beta cells 167 (135 -200) 208 (185 -232) 0. 043 4 (0. 2 -7) 40 (18 -62) <0. 001 205 (131 -279) 392 (323 -461) 0. 001 49 (30 -67) 69 (56 -81) 0. 095 Endocrine 49 (38 -60) 36 (31 -41) 0. 028 Exocrine 0. 7 (0. 2 -1. 4) 4. 7 (2. 4 -7. 0) 0. 003 Duct 41 (31 -50) 51 (46 -55) 0. 058 Damaged 10 (7 -12) 9 (8 -10) 0. 590 Number of recipients Beta cell per kg body weight (10 6/kg BW) P-value Graft cell number (x 10 6) Exocrine cells Duct cells Damaged cells Graft Composition (%)

Conclusions Duration of culture time correlates with clinical outcome after islet transplantation Human islet isolates with insufficient beta cell mass for implantation within 72 h can be cultured for 96 h and longer to combine preparations that together reach the set dose and result in metabolic benefit. The better outcome of grafts with a longer culture history was correlated to their higher endocrine purity, which is in line with observations in animal models.

Collaboration Beta Cell Bank Zhidong Ling’s Team UZ Brussel Bart Keymeulen Surgical Team: Daniel Tulleneers Eurotransplant (Belgian) Donor Centers Clinical Follow-up Teams Trial Centers BDR Centers Beta Cell NV Diabetes Research Center Danny Pipeleer’s Team Clinical Trials Pieter Gillard Da. Hae Lee Central Coordination : Ursule Van de Velde Team Immune Monitoring Frans Gorus Team Bart Roep Team