Haploidentical Transplantation Helen Heslop

Haploidentical Transplantation Helen Heslop

Outline Haplodentical transplantation Indications Regimens Outcomes KIR mismatching and NK cells Reconstituting T cell immunity Allodepleted T cells Cytotoxic T cells

Haploidentical Transplants

Options for Patients without a Matched Sibling Donor Unrelated donor? Degree of mismatch accepted Cord blood donor? Degree of mismatch accepted Haploidentical donor

Haplotypes Father 4/6 Mother 4/6 A1 A2 A2 A1 B8 B44 B7 B57 DR3 DR4 DR2 DR11 A1 A2 A1 A1 A2 A2 A2 A1 B8 B7 B8 B57 B44 B7 B44 B57 DR3 DR2 DR3 DR11 DR4 DR2 DR4 DR11 Patient Sibling 3/6 Sibling 3/6 Sibling 2/6

Haplotypes Husband Patient A1 A2 A2 A1 B8 B44 B7 B57 DR3 DR4 DR2 DR11 A1 A2 A1 A1 A2 A2 A2 A1 B8 B7 B8 B57 B44 B7 B44 B57 DR3 DR2 DR3 DR11 DR4 DR2 DR4 DR11 Child 4/6 Child 3/6 Child 3/6 Child 4/6

Haploidentical versus Unrelated Donor Advantages More rapidly available Donor available for additional products More graft versus tumor effects Disadvantages Increased alloreactivity Increased risk rejection and GVHD Delayed immune reconstitution

Haploidentical versus Cord Donor Advantages Donor available for additional products More graft versus tumor effects Less issues with cell dose of product Disadvantages Increased alloreactivity Increased risk rejection and GVHD

Algorithm for Donor Choices Published data Dependent on center aspects Regimens Research interests Experience and results with specific products Should be regularly reviewed

Sample Algorithm for Donor Choices HLA identical sibling 6/6 relative 5/6 matched relative 6/6 matched MUD or cord 5/6 matched cord 5/6 matched MUD, 4/6 matched cord or 4/6 matched relative 3/6 matched relative 3/6 matched cord

Limitations of Haploidentical Transplant Increased alloreactivity Initial study at Marsden using unmanipulated marrow after TBI/Cytoxan 10/35 with graft failure 12/35 died of hyperacute GVHD

Limitations of Haploidentical Transplant Increased alloreactivity Seattle studies showed equivalent outcome for 5/6 donors Increased GVHD but also reduced relapse Poor outcomes for 3/6 and 4/6 donors with standard regimens

Haploidentical Regimens Intensive Immunosuppression T cell depletion Anergization Megadose CD34-selected cells

Intensive Immunosuppression 135 patients received busulfan, cytarabine, cyclophosphamide, rabbit ATG Unmanipulated bone marrow and/or PBSC transplantation. GVHD prophylaxis with MMF, cyclosporine, and methotrexate. Grades II to IV acute GVHD 40% Two year leukemia-free and overall survival probabilities were 64% and 71% Lu et al

Intensive Immunosuppression Low-dose TBI, fludarabine +/- cyclophosphamide conditioning, and high dose cyclophosphamide on day + 3 post-transplant. 54% Grade II-IV GVHD 38% DFS O Donnell et al

T Cell Depletion Partial antibody-mediated T-cell depletion and post-transplant cyclosporine T10B9 (anti-alpha beta T-cell receptor) OKT3 (anti-cd3) Engraftment in >90% Grade II-IV GVHD 13% Long term DFS 20% in high risk patients Henslee Downey et al

Anergized Haploidentical BMT Induction of alloantigen-specific anergy by the co-culturing of host and donor bone marrow mononuclear cells in the presence of CTLA-4-IG 3/12 patients developed GVHD 5/12 survived 5-29 months Guinan et al

Megadose CD34-selected cells GCSF stimulation donor followed by CD34 selection 3-4 log T cell depletion High engraftment (>95%) Low GVHD (<10%) 48% DFS Aversa et al

Haploidentical Nonmyeloablative HCT Conditioning with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of alemtuzumab treated PBSC. GVHD prophylaxis with MMF and cyclosporine. 16% developed grade II-IV acute GVHD 6% primary graft failure DFS at 1 year 31% Rizzieri et al

Outcome of CD34 Selected Haploidentical Transplants 100 Censored 100 Death/Relapse 80 80 Survival 60 40 60 40 46% 20 20 0 0 1 2 3 4 5 6 12 18 24 30 36 42 48 54 60 Years post transplant

Causes of Failure Relapse 21% Infection 21% Regimen related mortality 5%

Causes of Delayed Immune Recovery Degree of HLA disparity Low numbers of T-cells infused Use of T-cell depleting antibodies Intensity of the conditioning regimen Impaired thymic function

Current Research Strategies Reduce Alloreactivity T reg cells Mesenchymal cells Reconstitute Immunity NK cells T cells

NK Cells and KIR matching

NK Cells Natural killer cells are a unique CD56+CD3- cell population Innate antiviral and antitumor immune response Different cell subsets with distinct phenotypic and functional characteristics 90% are highly cytotoxic CD56dim cells 10% are immunoregulatory CD56 bright cells NK cells recover as early as 2 to 3 weeks post-transplant by rapid differentiation from engrafted CD34+ cells

NK Cell Subsets CD16 CD56 CD16 CD56 IL-2R αβγ IL-2R βγ c-kit CD56 bright NK cell L-selectin CD56 dim NK cell CD94/NKG2A IFN-γ, TNF-α, GM-CSF, IL-10 KIR ADCC Natural cytotoxicity Immunoregulatory function Cytolytic function

(Killer Ig-like Receptors)KIRs Means by which NK cells recognize self from non-self 15 KIRs currenlty identified on chromosome 19 Ligand specificity for HLA molecules particularly HLA-C Some are activating and some are inhibitory

(Killer Ig-like Receptors)KIRs NK cells are activated by the absence of self MHC class I molecules on the surface of target cells. Expression of self MHC molecules on target cells delivers an inhibitory signal to NK cells via inhibitory KIRs. killing is inhibited, Killing of target cells occurs when NK cell inhibitory receptors are not engaged due to the absence of MHC class I due to MHC mismatch.

Other NK Receptors Activating receptors such as NKG2D and NKp46 trigger NK cell alloreactivity when engaged by antigens on virally infected cells and tumor cells NK cell activity is regulated by quantitative differences in cumulative inhibitory and activating signals transmitted via KIRs. presence or absence of the respective ligands on recipient cells determines if NK cells will be primed to be alloreactive and kill the targets

NK Cytotoxicity Balance of activating and inhibitory signaling NK Cell NK Cell Activating receptor Inhibitory receptor Activating receptor Inhibitory receptor Activating ligand HLA class I ligand Activating ligand Target Cell Target Cell

KIR Mismatch If donor NK cells are not fully inhibited by recipient MHC class I ligands there is graft NK cell alloreactivity KIR ligand mismatching in the GVH direction following T-cell depleted haploidentical HCT y Reduced relapse in patients with AML Lower rate of graft rejection (donor NK-mediated lysis of host residual T-cells) Reduction in GVHD (donor NK-mediated depletion of host antigen presenting cells) Ruggieri et al

Killing with KIR Mismatch Resistance Susceptibility Donor NK Cell Donor NK Cell Activating receptor Inhibitory KIR2DL1 (group 2 specific) Activating receptor Inhibitory KIR2DL1 (group 2 specific) Activating ligand HLA-Cw4 (group2) Activating ligand HLA-Cw3 (group1) Host Leukemic Blast Host Leukemic Blast

NK Alloreactivity NK Cell No Lysis X Autologous Normal Cell NCR p30 p44 p46 NKG2D CD94/NKG2A KIRs + + HA? MICA, MICB, ULBPs HLA-E HLA-A, -B, -C Donor NK Lysis Allogeneic Leukemia NCR p30 p44 p46 NKG2D CD94/NKG2A KIRs + + v Stress ligands v MHC ligands v Incompatible MHC

Anti-AML effects of NK cells Haplo-identical SCT (Ruggeri et al) MUD SCT (Giebel et al) MSD SCT (Hsu et al) Non-SCT (Miller et al)

Relapse after Transplant for AML Ruggeri et al 2007 Perugia

DFS in patients with AML

Effects in ALL Not seen in Perugia series Seen in St Jude study (Leung et al) repertoire assessment for the four KIRs by genotyping vs phenotyping was not in complete agreement

Exploiting NK Alloreactivity Donor selection criteria to include KIR genotyping Ex vivo expansion of NK cells for adoptive transfer Antibody blockade of inhibitory receptors

Blocking Recognition of Self Donor NK Cell Donor NK Cell Activating receptor Activating ligand Inhibitory receptor HLA-C Activating receptor Activating ligand Y Inhibitory receptor Anti-KIR antibody HLA-C Host Leukemic Blast Host Leukemic Blast

Adoptive Transfer Of Haploidentical NK Cells NK cell infusions given to 43 patients with refractory or relapsed malignancies NK cells prepared by pheresis, CD3 depletion, and IL-2 activation 40% NK cells 25% monocytes 20% B cells 1% T cells Established safe NK dose --Escalated dose from 10 5 to 2 x 10 7 cells/kg --No infusion reactions and no GVHD Miller et al Blood 2005

Natural Killer Cell Infusions Donor cell Recipient Cell Reduced Risk of NK Dendritic cell GVHD T-cell Rejection Viral infected cell Infection Leukemia cell Relapse

Adoptive Transfer of Purified CD56+ Cells Feasible to obtain on immunomagnetic column with CD3 depletion followed by CD56 selection Retain proliferative capacity Trials underway to study safety and feasibility in HSCT and non-hsct areas Leung et al J Immunol 2005

Reconstituting T Cell Immunity

T cell Reconstitution Pathways Early reconstitution via homeostatic peripheral expansion expansion of mature T-cells that survive the conditioning Expansion of mature T cells in the allograft Late reconstitution via a thymicdependent pathway.

Approaches to Reconstituting Immunity T cell precursor frequencies in haploidentical transplantation: alloreactive > anti-viral > anti-tumor Options Virus Specific T cells Allodepleted T cells

Cytotoxic T cells Anti-viral CMV EBV Adenovirus Anti-tumor Viral antigens -LMP1 and 2 antigens Chimeric-antigen-receptor transduced CTLs targeting GD2 or CD19

Generating antigen specific cytotoxic T cells ex vivo Repeated stimulation with antigen expressed on antigen presenting cell Expand antigen specific T cells T cells with specificities for other antigens will nor survive

Generation Of Antigen-specific CTL Antigen APC CTL specific for target antigen PBMC IL2 After 3 rd or 4 th stimulation analyze CTL lines ---> Freeze & QA/QC

Requirements for Generation CTLs Good antigen presenting cell Dendritic cells Monocytes B cells Source of antigen Viral lysate Transduce with full length viral antigen(s) Pulse with peptides Helpful to have immune donor

Studies with EBV and CMV specific CTL Studies Transferred CTLs specific for latent virus expand and persist long term Efficacy in preventing and treating CMV and EBV infection But Only target 1 virus Time for generation

Extending EBV Immunotherapy Approach to CMV and Adenovirus Choosing optimal antigen presenting cell Monocyte LCL (includes EBV antigens) Source of CMV and adenovirus antigen? -Ad5f35 vector encoding pp65? Competition between viral antigens

Generation Of Adv-specific CTL Using Ad5f35 Vectors LMP1 LMP2 pp65 EBNA 3a,3b,3c LP EBNA-2 EBNA-1 Trivirus Bivirus CTL CTL lines lines (Adv/EBV/CMVpp65) (Adv/EBV) Lymphoblastoid cell lines (LCL)

Generation Of Virus-specific CTL Ad5f35 vector PBMC Ad5f35 vector actmo LCL EBV-LCL + IL-2 O/N Adherence Activated monocytes + PBMC Virusspecific CTL After 3 rd or 4 th stimulation analyze CTL lines ---> Freeze & QA/QC

Analysis of CTL lines 34 CTL lines analyzed - 19 Bi-virus specific - 15 Tri-virus specific (CMV seropositive donors) Phenotypic analysis Specificity for viruses: - Cytotoxicity assay - ELISPOT assay for IFN-γ secretion - Pentamer analysis

CTL Phenotype % % cells 100 100% 80 80% 60 60% 40 40% 20 20% 0 0% CD4 CD8 CD56 CD19 CD14 CD4 CD8 CD56 CD19 CD14 Bi-virus specific CTL lines % 100 80 60 40 20 0 CD4 CD8 CD56 CD19 CD14 Tri-virus specific CTL lines

CTL Specificity-Cytotoxicity CMV target EBV target Adv target Allo target Bivirus-specific CTL Mean of 19 lines Trivirus-specific CTL Mean of 15 lines

CTL Specificity-IFNγ ELISPOT Analysis CTL lines Adv EBV CMV CONTROL SFC / 1 x10 5 cells Bi-virus median (range) 558 (47-1578) 414 (146-976) n/a 10 (1-32) Tri-virus median (range) 86 (20-350) 183 (46-351) 648 (181-1278) 14.5 (3-65) 19/19 Bi-virus & 14/15 Tri-virus specific CTL were Adv specific All CTL lines were EBV specific All Tri-virus-specific CTL were CMV specific

Antigenic competition Bivirus Trivirus Pool 1 (aa 1-100) Pool 2 (aa 86-185) Pool 3 (aa 171-270) Pool 4 (aa 256-355) Pool 5 (aa 341-440) Pool 6 (aa 426-525) Pool 7 (aa 511-610) Pool 8 (aa 596-695) Pool 9 (aa 681-780) Pool 10 (aa 766-865) Hexon peptide pools Pool 11 (aa 851-952) Pool 1 (aa 1-100) Pool 2 (aa 86-185) Pool 3 (aa 171-270) Pool 4 (aa 256-355) Pool 5 (aa 341-440) Pool 6 (aa 426-525) Pool 7 (aa 511-610) Pool 8 (aa 596-695) Pool 9 (aa 681-780) Pool 10 (aa 766-865) Hexon peptide pools Pool 11 (aa 851-952) CD8 epitope CD4 epitope Pt 14 Pt 15 Pt 16 Pt 17 Pt 18 Pt 19 Pt 20 Pt 21 Pt 22 Pt 23 Pt 24 Pt 25 Pt 26 Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6 Pt 7 Pt 8 Pt 9 Pt 10 Pt 11 Pt 12 Pt 13 Greather breadth of Adv reactivity in Bivirus-specific CTL lines

Eligibility Criteria CMV prophylaxis and Adenovirus prophylaxis and treatment studies Day +30 post HSCT GVHD <grade III at enrollment

Infused CTL lines 27 CTL lines infused -25 on-study -2 compassionate CTL dose range: 5x10 6 to 1.35x10 8 /m 2 12 patients received Bi-virus CTL 15 patients received Tri-virus CTL

Patient Characteristics Group Median age (range) Alternative donors Campath or ATG in vivo Median day CTL infused Off immune suppression Bivirus 4yr (1-13) 100% (13/13) 100% (13/13) +77 (22-150) 61% (8/13) Trivirus 10yr (1-62) 73% (11/15) 87% (13/15) +84 (35-164) 60% (9/15) 79% of patients received BMT for malignant disease 4 patients who received Bivirus CTL had Adv 5 patients who received Trivirus CTL had Adv

Toxicity No dose limiting toxicity

Efficacy of EBV and CMV -specific CTL Infusion After HSCT Mean SFC per 1x10e6 cells Nat Med. 2006;12(10):1160-1166 EBV and CMV-specific- T cells expanded after infusion and were protective in vivo 18050 160 140 120 30 100 8020 60 4010 20 SFC/1x10e6 cells 0 Infected Uninfected pre 1wk 2wk 4wk Pre 1 2 4 weeks Adv Mean SFC per 2x10e5 cells Mean SFC per 2x10e5 cells 180 160 140 120 100 80 60 40 20 0 180 160 140 120 100 80 60 40 20 0 Pre 1 2 4 weeks Pre 1 2 4 weeks EBV CMV

Clinical Outcome Summary-CMV 7/15 patients developed CMV reactivation immediately pre or post CTL infusion 6/7 had decrease in CMV viral load with corresponding elevation in CMV-specific CTL detected in PB 6/7 cleared virus long term (6-33mths) 1/7 resistant to antiviral Rx

Clinical Outcome Summary-EBV 8/28 patients had EBV reactivation All patients had decrease in EBV viral load with corresponding elevation in EBV-specific CTL detected in PB No antiviral therapy required

Resolution of Liver Lesion No Further Therapy Required Diagnosis of PTLD 2 months later

Reduced Adv load in blood and stool post- CTL Adv copies/ml blood 4000 3000 2000 1000 Adv copies per ml blood Adv copies/gram stool x10e6 1600 1200 800 400 Adv copies/g stool 0 0 pre 2 4 8 12 weeks 3yr old female; Improvement of diarrhea 2wks post-ctl

Patient with Adenovirus Disease Admitted for pneumonia/hypoxia Day +133 Day +138 BAL positive for adenovirus Required HFOV, inotropes, started Cidofovir

Resolution of Adenoviral Pneumonia after CTL Day +148 5x10 6 adenovirus-specific CTLs Day +166 extubated successfully

Reduction in Adv Load Post-CTL & Rise in Adv CTLp SFC per 1 x 10 6 cells 300 250 200 150 100 50 Adv T cell Adv DNA (trachea) 1,400 1,200 1,000 800 600 400 200 Adv copies/ml 0 Pre CTL 2 wk 4 wk 6 wk 8 wk 0

Summary Bivirus-specific CTL routinely generated and have greater and broader adenovirus activity than trivirus specific CTL Ad-specific CTL expand only in presence of Ad infection Role of Ag in homeostatic expansion Expansion and clinical activity not related to dose level Leen et al Nat Med 2006

Future Directions Expand the number of antigens targeted Aspergillus RSV Influenza pp65 Parainfluenza BK virus Herpesvirues Combine immunotherapy with vaccination strategies

How Do We Extend Applicability? Simplify production patient specific product Modify APC Early selection Can we use bank of allogeneic matched CTLs

Alternative Rapid Selection Procedures Select low frequency antigen-specific CTLs Transfer small numbers selected cells Rely on expansion in vivo

Methods to Select T-cells tetramer CTL IFN-γ IFN-γ Tetramer selection Gamma interferon selection

Rapidly Available CTLs All have clinical activity Allogeneic banked cells Risk of rejection Tetramer selected cells Restricted specificity Gamma interferon capture selected cells Large volumes blood needed

Allodepletion Activated T cells express CD25, CD69, CD71 and HLA-DR If stimulate donor T-cells with host can remove responding T-cells with antibody

Selective Depletion Of Alloreactive Cells + 72 hours Murine IgG1 anti-cd25 conjugated to deglycosylated ricin A chain Donor PBMC Recipient EBVlymphoblastoid cell line (Irradiated) Alloreactive cells upregulate CD25 Allodepleted T-cells Amrolia at al Blood 2006

Patients 17 patients Median age 8 years (range 2-58) Median follow up 30 months Dose level 1 (9 patients): 10 4 /kg x3 Dose level 2: (8 patients) 10 5 /kg x3 6 not in remission 5 patients relapsed post previous transplant

CD3 Recovery Post HSCT Months post transplant 1800 1600 1400 1200 1000 800 600 400 200 0 Dose Level 2 -- Dose Level 1 -- 1 2 3 4 5 6

Reconstitution Immunity to EBV SFC/10E6 PBM 4000 3500 3000 2500 2000 1500 1000 500 0 Dose Level 1 Dose Level 2 4000 3500 3000 2500 2000 1500 1000 500 0 1 2 3 4 5 6 7 8 9 1 1 2 3 4 5 6 7 8 9 1 Months Post HSCT

Recovery Of CD8 Response To CMV Donor A2-NLV 0.4% B8-ELR 0% Patient At 6 months 5.7% 3.9% Response larger and to additional epitopes compared to donor

Recovery From PML 14 year old with MDS 2 months post haplo SCT presented with tremor, inhibition and progressive LOC 2 months post HSCT CD3 and 4 = 0/mm 3 4months post HSCT CD3=630/mm 3, CD4=220/mm 3

GVHD Acute GVHD 2/17 1 grade 2 skin 1grade 4 skin Chronic GVHD 2/14 1 ext. skin/mouth 1 ext. liver

Clinical Status 6/14 Baylor patients alive in CR 24-52 months post allodepleted cells 6 patients relapsed

Conclusions Adoptive immunotherapy with allo-depleted donor T-cells More rapid recovery of T-cells Effector memory phenotype Accelerated recovery of viral-specific immunity with clinical responses Low incidence of GVHD and viral deaths Significant relapse risk

Future Directions Dose escalate further to evaluate effect on risk of relapse Risk of GVHD Transition to allodepleted T cells modified with retroviral vector encoding icaspase9 and CD19

Can we extend approach to antitumor immunity? Antigens less well defined Antigens less immunogenic Other tumor evasion mechanisms

Extending CTL Therapy to Type II latency EBV tumors EBV infected B cell EBNA 2 EBNA 3 LMP1 EBNA 1 LMP2 BZLF1 T T T T T T Lytic EBNA 3 LMP 2 LP LMP 1 EBNA 2 EBNA1 Hodgkin s Or NPC tumor cell EBNA 1 LMP LMP1 1 LMP 2 Inhibitory cytokines

Making LMP2 an Immunodominant Antigen Ad5f35 LMP2 DC LCL IL-2 IL-2 IL-2 PBMC LMP2- specific CTL Gahn et al Int J Cancer 2002, Bollard et al, J Immunother 2004

LMP2-specificity of LMP2-CTL Line Generated From Patient With Relapsed Hodgkin Disease Tet-CLG Tet-FLY FC125Y04.010 10 0 10 1 10 2 10 3 10 4 CD8 FITC FC125Y04.011 26.4% 13.5% % specific lysis 70 60 50 40 30 20 10 Allo LCL Auto LCL PHA blasts Fibros GFP Fibros LMP2 10 0 10 1 10 2 10 3 10 4 CD8 FITC 0 40 to1 20 to 1 10 to 1 5 to 1

Clinical Results Relapsed Disease Arm No toxicity 1 progressive disease 1 very good partial response >18 months 4 complete responses Persisted 9 to 36+ months

Complete Radiological Response EBV+ve NK-T NHL Pre CTL Post CTL E B V c o p i e s / u g D N A 7000 6000 5000 4000 3000 2000 1000 0 Pre CTL 1 Pre CTL 2 Pre CTL 3 pre CT L1 1 wk 2 w ks/prectl 2 4 wk s 6wks prect L3 1wk 2 wk

Immunohistochemistry L Carytenoid Pre CTL Post CTL EBER 10x CD4 40x

Conclusions LMP2 specific CTLs have activity in EBV+ve Hodgkin s Disease and Type II latency NHL 4/6 CRs in patients with active disease (autologous) Bollard et al Blood 2007

Can We Extend Beyond Viral Antigens? Many express specific surface markers GD2 CD19 CD30 kappa Prepare chimeric T cells with antibody specificity See whole antigen on tumor surface

Improving T cell Therapies Grafting Additional Specificities α β TcR-complex γε εδ ζζ Fab v H v L Antibody C L C H1 C L C H1 v L v H v H v L

Overcoming limitations of CARs Dual-specific T cells -On EBV-CTL (Rossig et al, Blood 2002) -On Allo T cells (Kershaw et al, Nat Biotech 2002) -On MP-1-specific CTL (Cooper et al, Blood 2004) EBV-Infected B cell CD4+EBV CTL CD8+EBV CTL Cognate help for CTL activation and expansion tumor

Are CAR-CTL better than CAR-PTC in Neuroblastoma Patients? Transduce patient PTC and CTL with a vector encoding identical receptor but distinct oligonucleotide for each population 11 patients with relapsed neuroblastoma

Percent Transduced T cells in Blood 0.2 0.15 % 0.1 0.05 GD2 PTC 0 1 1Wk 2Wks 4Wks 6Wks Time post-infusion

Percent Transduced T cells in Blood 0.2 0.15 % 0.1 0.05 GD2 CTL GD2 PTC 0 1 1Wk 2Wks 4Wks 6Wks Time post-infusion

Dose 1 Complete Response Pre-Infusion 2 Months 4 Months

Engineering Haploidentical Grafts