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Treg adoptive immunotherapy in full-haplotype mismatched HSCT

Treg adoptive immunotherapy in full-haplotype mismatched HSCT. Massimo Fabrizio Martelli. Hematology and Clinical Immunology Section University of Perugia, Italy. Conditioning. Median Dose of CD34 + Cells 12,8x10 6 /kg b.w. Median Dose of CD3 + Cells 1x10 4 /kg b.w.

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Treg adoptive immunotherapy in full-haplotype mismatched HSCT

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  1. Treg adoptive immunotherapy in full-haplotype mismatched HSCT Massimo Fabrizio Martelli Hematology and Clinical Immunology Section University of Perugia, Italy

  2. Conditioning Median Dose of CD34+Cells 12,8x106/kg b.w. Median Dose of CD3+ Cells 1x104/kg b.w. Median Dose of CD20+ Cells 4.1x104/kg b.w. 8 Gy TBI in a single fraction at 16 cGy/m Thiotepa ATG Fludarabine Reisner Y and Martelli MF: Immunol.Today 1995, 16, 437

  3. Engraftment GvHD No of patients=196 92% 98% No post-transplant immunosuppression 2% 3% Primary Overall Acute Chronic Aversa F. et al., N Engl J Med 1998;339:1186-1193 Aversa F. et al., J Cln Oncol 2005;23:3447-3454

  4. Post-transplant generation of alloreactive NK repertoire (Ruggeri L., Velardi A. Blood 1999) NK repertoire Host targets HLA-C group 1 Cw2/Cw4 High-intensity conditioning KIR2DL1 Missing self KIR2DL 2/3 HLA-Bw4 KIR3DL1 Stem Stem Stem 600 Tcell Stem Stem Stem Stem Stem Stem CD16 400 Stem cells/cmm Stem CD8 200 CD4 0 Mega-dose stem cells from “allo NK” donor No post-transplant immune suppression 0 50 100 150 200 The reconstituting NK cells have the same repertoire as the donor, including high frequency of donor versus recipient alloreactive NK clones A potentially NK alloreactive donor occurs in nearly 50% of transplant pairs

  5. 1,0 0,8 0,6 Non-NK alloreactive (n=32) 0,4 P = 0.03 0,2 NK alloreactive (n=32) 0,0 0 5 10 15 20 Years Post-transplant leukemia relapse in T-cell depleted haplo HSCT 64 AML Patients (CR I #24, CR II #28, CR≥III #12 ) Cumul. incidence of relapse Ruggeri et al., Science 2002; Blood 2007; Stern et al., Blood 2008; updated 2011

  6. 0.48 CR 1 CR ≥ 2 EBMT-ALWP survey Ciceri et al. Blood 2008 Disease free survival in high-risk AML T-cell depleted haplo HSCT T-cell depleted haplo HSCT 0.50± 0.09 CR 1 (n=34) Prob of DFS Prob of DFS 0.35± 0.07 CR ≥ 2 (n=49) Aversa F. et al., N Engl J Med 1998 Aversa F. et al., J Cln Oncol. 2005

  7. High-risk AML Diagnosis and HLA-typing No HLA-identical sibling Search for alternative donors the perplexing question: which is the best option? URD, partially matched No well-matched URD UCB unit Haplo family member

  8. Post-transplant immune recovery in T-cell depleted full haplotype mismatched HSCT Infection 66% Very narrow T- cell repertoire • Few T- cells in the graft so as to prevent GvHD • Extra in vivo T- cell depletion by ATG which also • antagonizes T-cell homeostatic expansion Cumulative incidence of TRM 0.36 for 145 AL patients in any CR Others Rejection CNS toxicity Fatal Infections MOF GvHD I.P.

  9. anti CMV or anti Aspergillus CD4+ clones after screening for cross-reactivity to host alloantigens Improving post-transplant immunity after HLA-haploidentical HSCT Adding back mature donor T-cells that are pathogen specific a) suicide gene insertion (i.e. HSV-tk) into T-cells allows switch-off of GvHD donor T cells ex vivo depleted of anti-host alloreactivity ( i.e. photodynamic purging of alloreactive T cells; depletion of activated T cells with anti-CD25 bound to beads) b) anti EBV Adding back mature donor T-cells with a broad repertoire Clinical techniques to prevent graft versus host disease a) b)

  10. Rebuilding post-transplant immunity Lessons from animal models Adoptive transfer of naturally arising CD4+CD25+ regulatory T cells (Tregs), when coinfused with conventional T lymphocytes (Tcons), prevents GvHD, while favoring immune reconstitution Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses Sakaguchi et al., Ann Rev Immunol, 2004, 22:531-562 Donor type CD4+CD25+ regulatory T cells suppress lethal acute graft-versu-host disease after allogeneic bone marrow transplantation Hoffmann et al., J Exp Med, 2002, 196:389-399 CD4+CD25+ regulatory T cells preserve graft versus-tumor-activity while inhibiting graft-versus host disease after bone marrow transplantation Edinger et al., Nat Med 2003, 9:1144-1150 In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation Nguyen et al. Blood 2007, 109:2646-2656 The impact of regulatory T cells on T-cell immunity following hematopoietic cell transplantation Nguyen et al., Blood 2008, 111:945-953 Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered with regulatory T cells for GvHD prevention Gaidot et al. Blood 20010, 117:2975-2983

  11. Gate on CD4CD25+high Starting fraction Final fraction Cells (x109)1060 (540-1370) 280 (202- 390) %CD4CD253.0 (1.5-7.45) 92.4 (90-97.1) N° cells (x 106)330 (221-1020) 256 (185.6-365.4) %CD4CD25high0.3 (0.12- 0.89) 33.6 (14.4-39.6) N° cells (x 106)36.12 (19.98 - 84) 68.6 (20.9-143) Gate on CD4CD25+ CD25 CD4 FoxP3 CD127 Selection and Characterization of CD4+CD25+ Regulatory T Cells 1st step: Depletion of CD8+/CD19+cells Leukapheresis product Fully Automated Immunomagnetic Selection of CD4+CD25+Cells 2nd step: Enrichment of CD25+ cells Fox P3+ cells 71.9 ± 15 % Di Ianni M et al.Blood 2011; 117:3921-3928 Di Ianni M et al., Blood in press

  12. Starting fraction Final fraction Cells (x109)1060 (540-1370) 280 (202- 390) %CD4CD253.0 (1.5-7.45) 92.4 (90-97.1) N° cells (x 106)330 (221-1020) 256 (185.6-365.4) %CD4CD25high0.3 (0.12- 0.89) 33.6 (14.4-39.6) N° cells (x 106)36.12 (19.98 - 84) 68.6 (20.9-143) Selection and Characterization of CD4+CD25+ Regulatory T Cells 1st step: Depletion of CD8+/CD19+cells Leukapheresis product Fully Automated Immunomagnetic Selection of CD4+CD25+Cells 2nd step: Enrichment of CD25+ cells Final Cell Fraction Enhanced expression of CD62L, CD39, GITR, CTLA4 CD45RO was the predominant isoform while the CD45RA was around 10%.

  13. Harvesting Tregs from peripheral blood before stem cell collection increases cell number in starting fraction and significantly enhances the Treg count in the final fraction Post HSC collection Pre HSC collection Total n° Cells (x 106)*163.8 (58.2 - 387) %CD4CD25 88.7 (76.2 – 91.7) N° cells (x 106)* 149.9 (57.2 – 321) %CD4CD25high11.8 (0.9 – 25.5 )** N° cells (x 106)* 17.0 (0.7 - 41.8)** Total n° Cells (x 106)*280 (202 - 390) %CD4CD2592.4 (90 – 97.1) N° cells (x 106)*256 (185.6 – 365.4) %CD4CD25high33.6 (14.4 – 39.6)** N° cells (x 106)*68.6 (20.9 - 143)** N=8 N=15 * median+range ** p<0.05

  14. Tregs inhibit MLR in a dose dependent way

  15. Alloantigen APCs pre-incubation enhances Treg-mediated immunosuppression Freshly selected polyclonal T-reg cells were co-cultured in the presence of allogeneic APCs for 48 hours. Cells were plated under limiting dilution conditions and proliferating T cell clones counted.

  16. Treg and Tcon adoptive immunotherapy in haplo HSCT for patients with high risk AL 2x106/kg 10x106/kg Tregs CD34+ TBI based conditioning days No post-transplant immunosuppression 1st clinical trial Tcons Sept 2008-Oct 2009 8 Gy TBI Thiotepa Fludarabine Cyclophosphamide 1x106/kg Blood 2011; 117:3921 In vivo expansion of donor Tregs in the setting of HLA disparity Di Ianni M et al.

  17. PRIMARY ENGRAFTMENT 26/28 (93%) Grade ≥II 2/26 GRAFT vs HOST DISEASE The two cases of GvHD were among the 5 patients who had received 4x106 Kg/bw Tregs and 2x106 Kg/bw Tcons

  18. Pattern of immunoreconstitution Recovery of CD4+ and CD8+ T cell subpopulations Spectratyping Donors Complexity score Months after transplant

  19. Reconstitution of pathogen-specific T-cell repertoire Limiting dilution analyses of pathogen-specific CD4+ and CD8+ cells Standard Haplo (n=150) Haplo with T-reg (n=26) Proliferating CD4+ pathogen -specific T cells per 106 cells INF-g producing CD8+ pathogen -specific T cells per 106 cells Months after transplant

  20. T cell response to CMV: cytokine profiles Sbj IL-2 TNF-a IL-13 IFN-y IL-2 TNF-a IL-13 IFN-y VE-SE 1 GI-MO 17 RO-DO 20 LE-AN 23 BA-SI 24 CD8+ CD4+

  21. Reconstitution ofperipheral blood B cell number, B cell repertoire and seric levels of immunoglobulins Fr1-JH Fr1-JH Fr2-JH Fr2-JH Fr3-JH Fr3-JH Day + 30 post BMT Day + 90 post BMT IgG IgA IgM 3 months 733 (80-1530) 31 (1-72) 71 (17-229) 63 (45-140) 6 months 692 (411-876) 44(8-111)

  22. Are these patients immunologically competent ? In accordance with ISS guidelines 7 subjects ( ≥ 3 months after stem cell transplantation) were vaccinated against pandemic influenza with 2 doses of MF59-H1N1california. No vaccination for seasonal flu. Day 0 30 60 240 visit 1 2 3 4 6 months Hemoagglutinin Inhibition Assay (HI) HI titer ≥1:40 = protection against H1N1 HI ≥ 4 fold = vaccination efficacy

  23. Profile over time of Flu specific CD4+ after MF59-H1N1california vaccination 0.1 pre immune 30 days post 1 dose 30 days post 2 dose 0.08 0.06 % Ctk+ CD4+ / CD4+ T cells 0.04 0.02 0 Patient No. 6 11 17 20 23 24

  24. Rebuilding post-transplant immunity Adoptive immunotherapy with naturally occurring polyclonalTregs controls alloreactivity of a significant number of coinfused Tcons favors immune reconstitution since alloantigen-specific Tregs do not cross-inhibit pathogen- specific Tcon responses

  25. TRM 1.0 1.0 0.8 0.8 0.6 0.6 Probability 0.4 0.4 0.2 0.2 0.0 0 1 2 3 4 0.0 Years 0 1 2 3 4 5 Years Tregs and Tcons in haplo HSCT for patients with high risk AL Disease Free Survival Veno-occlusive disease (3) Multi-organ failure(1) Adenoviral infection (1) Adenoviral infection and GvHD (1) GvHD (1) Bacterial sepsis (1) Systemic toxoplasmosis (1) Fungal pneumonia (4) CNS aspergillosis (1) Median follow up:35 months (range 32 – 43) Updated 16-03-2012

  26. Treg and Tcon adoptive immunotherapy in haplo HSCT for patients with high risk AL 2x106/kg 10x106/kg T regs CD34+ TBI based conditioning days No post-transplant immunosuppression 2nd clinical trial 1st clinical trial May 2010-Oct 2011 Tcons Sept 2008-Oct 2009 Alemtuzumab (20mg/sqm; day-21) 8 Gy TBI Thiotepa Fludarabine 8 Gy TBI Thiotepa Fludarabine Cyclophosphamide 1x106/kg Blood 2011; 117:3921 In vivo expansion of donor Tregs in the setting of HLA disparity ASH 2011 Di Ianni M et al.

  27. PRIMARY ENGRAFTMENT 23/23 (100%) Days Days Grade ≥II 4/23 (17%) GRAFT vs HOST DISEASE 2/4 patients resolved GvHD, 2/4 patients died of GvHD

  28. 1.0 0.8 1,0 CTX Prot Alemtuzumab Prot 0,8 0.6 0,6 Probability 0.4 0,4 0,2 0.2 0,0 FU 0 10 20 30 40 0.0 0 1 2 3 4 5 Years Tregs and Tcons in haplo HSCT for patients with high risk AL DFS TRM Fulminant Hepatitis (1) GvHD (2) HHV6 pneumonia (1) HHV6 disease (1) Fungal Pneumonia (3) Fusariosis (1) Years Median follow up:19 months (range 11 – 24) Updated 16-03-2012

  29. 1.0 1.0 0.8 Alemtuzumab Lot #84039D (9 pt) 0.8 Alemtuzumab Lot #84039D Alemtuzumab Lot #F8003H01(14 pt) 0.6 Alemtuzumab Lot #F8003H0 0.6 0.4 Probability 0.4 0.2 0.2 0.0 0 1 2 3 4 5 0.0 0 1 2 3 4 Years Tregs and Tcons in haplo HSCT for patients with high risk AL TRM DFS Years

  30. Pattern of immunoreconstitution 1st Alemtuzumab lot (# 84039D) Recovery of CD4+ and CD8+ T cell subpopulations days after transplant days after transplant >50 >100 >200 >50 >100 >200 CD8/mmc CD4/mmc Within 100 days, mean values of CD4+ and CD8+ T cells are more than 200/mmc

  31. Treg and Tcon adoptive immunotherapy in haplo HSCT for patients with high risk AL 2x106/kg 10x106/kg T regs CD34+ TBI based conditioning days No post-transplant immunosuppression Jan 2012 Thymoglobuline (7.5mg/kg; day -21) 8 Gy TBI Thiotepa Fludarabine Tcons 1x106/kg Gancyclovir Foscarnet Ambisome ESA pentanoic acid

  32. Treg and Tcon adoptive immunotherapy in full haplotype mismatched HSCT Post-TransplantLeukaemia Relapse • Demographics: • 25 patients (NK allo 11/25): • 22 AML (NK allo 10/22) • 1 ALL • 2 Biphenotypic AL 2/2 with FLT3-ITD+, NMPc+ AML 1/2 in molecular relapse at transplant 2/2 transplanted from a non-NK allo donor Relapse: 2/25 Median follow up:20 months (range 8 – 41) Updated 01-03-2012

  33. How is the GvL effect maintained? Treg immunotherapy did not impact upon post-transplant generation of donor vs recipient alloreactive NK cell clones High number of infused Tcons No post-transplant immunosuppression

  34. Translational Research in Full-Haplotype Mismatched Transplant HEMATOLOGY AND IMMUNOLOGY SECTION DEPARTMENT OF IMMUNOLOGY UNIVERSITY OF PERUGIA WEIZMANN INSTITUTE OF SCIENCE Head:Prof Brunangelo Falini Head: Prof Yair Reisner BMT Unit Franco Aversa Alessandra Carotti Adelmo Terenzi Rita Felicini Antonio Pierini Valentina Zoi Cinzia De Fazio Maria Speranza Massei Immunology Andrea Velardi Loredana Ruggeri Katia Perruccio Elena Urbani Antonella Tosti Graft Processing Franca Falzetti Mauro Di Ianni Paolo Sportoletti Tiziana Zei Roberta Iacucci Elisabetta Bonifacio Beatrice Del Papa Debora Cecchini Alain Bell Flora Castellino Chimera of Arezzo c.400 BC A votive bronze dedicated to the supreme Etruscan God of day, Tin orTina

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