Third Party Cord Blood Derived Fucosylated Regulatory T cell Prevent

Graft versus Host Disease

Simrit Parmar, MD

Acute GVHD• Acute GVHD

– Typically occurs around the time of engraftment.– Previously mis-defined as GVHD which occurs prior to day

100 post-transplant.

– Three main organs involved:• Skin: macularpapular rash• GI system: Nausea / Vomiting and Diarrhea• Liver Abnormalities: typically cholestatic (jaundice).

– Incidence of 9-50% of sib transplants.

Vigorito et al. Blood 2009

Acute GVHD: Pathophysiology1. Recipient conditioning

2. Donor T cell activation

3. Cellular and Inflammatory Effectors

Approaches to the Prevention of GVHD

• Pharmacologic– CNI/MTX– CNI/MTX vs Rapa/MTX

• Graft source– BM vs PBPC– MRD vs URD vs UCB

• T Cell depletion– CD34 Selection– ATG, Campath

• Immune regulation

Regulation of Immune Function

• Critically important in health and disease• Compartmentalization of immune

responses• Cytokines• Regulatory T cells (Treg, NK-T, iTreg, others)

RegulationReactivity

T regulatory cell T effector cell

CD4+ T Cell Subsets

Magenau et al. BBMT. 2010.

38%

63%

Circulating Tregs predict OS

Day 0 Phenotype

Positive Selection

NegativeSelection

Expanded Cells show Treg Phenotype

Expanded UCB Tregs maintain Gaussian Distribution of TCR VβRepertoire

Expanded Tregs are Functional

Expanded UCB Tregs show FOXP3 Demethylation

Xenogenic GVHD Mouse Model

Lymphoid follicle

Extra-medullary hematopoeisis mixed with histiocytes

Spleen

Small Intestine 400x (Apoptosis with Enteritis)Apoptotic bodies diagnostic for GvHD (arrows)

Apoptotic bodies

Interstitial edema

Skin

vein B.D.

AB.D.

Yellow arrows represent dead hepatocytes. Outlined area is a combination of necrotic hepatocytes, fibrosis, and some lymphocytes. Scattered dark blue nuclei within outlined area are mostly lymphocytes. Liver 40X

Small IntestineLiver 40X

Firefly luciferase labeled CB Tregs were able to proliferate in response to stimulation by PBMC

3rd party CB Tregs prevent GVHD

Day -1UCB Treg 1x107

Day -1320 cGy

Day 0PBMC 1x107

CONTROL TREG PROPHYLAXIS

3rd party CB Tregs prevent GVHD

Phenotype Weight GVHD Score

3rd party CB Tregs prevent GVHD

Overall Survival Circulating Inflammatory Cytokines

3rd party CB Tregs prevent GVHD PBMC PBMC+Treg

Challenges for Clinical Translation of Treg

• Treg are rare cell populations

• Paucity of unique markers for isolation and availability of clinical grade reagents

• Marginal functional assays in humans

• Regulatory requirements

Can we improve the Efficacy of CB Tregs?

Fucosyltransferase (FT) VIHematopoieticprogenitorcell

FucoseGlycoprotein

Fucosylatedglycoprotein

YY

YY

Y√

X

YY

Y

P- & E-SelectinBM

endothelium

Impact onhoming &

engraftment?

YY

Bone marrowmicrovasculature

Enhancing selectin binding through ex vivo fucosylation

Fucosylation of CB CD34+ cells using FT-VI enhancesengraftment in NSG mice

4.5x104 CD34+ cells/mouse

Fucosylated

Untreated

100 101 102 103 104

CLA FITC

CB FTVI 40.008100 101 102 103 104

CLA FITC

washed cb.008

CLA

Untreated

Fucosylated

CD34

Does Fucosylation have effect on Treg homing?

Fucosylation efficiency of Treg

FTVI Treated

Biotin CLA Strep APCBiotin IgM Strep APC

UnTreated

Biotin CLA Strep APCBiotin IgM Strep APC

0.4% 8.8% 0.3% 62%

Fucosylated Tregs and Selectin Pathway Binding?

Fucosylation of Tregs leads to increased ability to bind E-selectin ligand

Figure 1D

Figure 1E

Homing pattern of fucosylated Treg vs non-fucosylated Treg

FTVI

CB

CD25 selection

CD25+ cells

eGFP-FFluc retro-viral transduction

Continually culture for 11 days

Treg expressing eGFP-FFluc

No FT-VI treatment FT-VI treatment

Treg FT-Treg

Experiment procedure

No FT-VI treatment

CB

CD25 selection

CD25+ cells

eGFP-FFluc retro-viral transduction

Continually culture for 11 days

Treg expressing eGFP-FFluc

FT-VI treatment

Treg FT-Treg

Experiment procedure

Day -2 Day -1 Day 0

XRT Treg/FT-Treg PBPC

cell dose: 107/mouse

Day -1

Day 0

Day 3

Day 4

Day 5

In vivo Bio-distribution of Fucosylated Tregs vs. Untreated Tregs

FTVI-TREG TREG

Treg: HLA-A2 negative/ GFP

PBPC: HLA-A2 positive

Fucosylated Tregs persist for longer duration in vivo vs. untreated Tregs

Day 7

Day 10

Day 12

FTVI-TREG TREG

Lesser number of GVHD causing CD45+ lymphocytes are present in Fucosylated Treg Recipients LNs

FT-TREG+PBPC TREG+PBPC

BM 10.4673 50.8491

Axillary LN 1.93 93.4065

Mesenteric LN 1.13 42.1731

Spleen 37.5158 94.4055

Inguinal LN 13.2 51.1461

Cervical LN 0 11.6

Are Fucosylated Tregs more Effective?

Fucosylated Tregs 1 x 10e6

Untreated Tregs 1 x 10e6

PBPC1 x 10e7

PBPC1 x 10e7

Day -2320 cGy

Day -1

Day 0

Monitor for weight, GVHD score and survival

Fucosylated Treg recipient maintain weight for longer duration

(Act

ua

l we

igh

t – b

ase

line

we

igh

t)/

bas

elin

e w

eig

ht

Fucosylated Treg Recipients have significantly longer survival compared to Untreated Tregs

Proposed Clinical Trial

Day -8 Day -7 Day -6 Day -5 Day -4 Day -1 Day 0 Flu 40mg/m2

Flu 40mg/m2

Flu 40mg/m2

Flu 40mg/m2

TBI Treg infusion

CB Infusion

Cy 50mg/kg + MESNA

Flu, fludarabine; Cy, cyclophosphamide

Fucosylated Tregs

Immune Prophylaxis with MMF + Sirolimus

41

Year 1 2 3

Quarter 1 2 3 4 1 2 3 4 1 2 3 4

Specific Aim 1  1. Validate clinical grade Treg expansion with CD3/28 beads in presence of interleukin-2 and subsequent fucosylation in the GMP laboratory

   

2. FDA IND application and IRB approval of the clinical protocol

3. Enrollment of first 5 patients onto the clinical protocol

4. Enrollment of the next cohort of 7 patients

5. Enrollment of the last cohort of 8 patients

6. Assess Engraftment and Chimerism Analysis

7. Perform immune correlatives and study GVHD biomarkers and circulating inflammatory cytokines

Specific Aim 2

1. Elucidate the homing pattern of fucosyltransferase-VI vs. VII treated Tregs in a xenogenic GVHD mouse model.2. Determine the role of the selectin ligands PSGL-1 in FT-VI vs. FT-VII-mediated CB Treg homing to the sites of inflammation in the NSG GVHD model. Manuscript 1: Third party Cord Blood Derived Ex Vivo Expanded and Fucosylated Regulatory T Cells Effectively Prevent GVHDManuscript 2: Saftey and Efficacy of Fucosylated Tregs in patients undergoing Double Cord Blood Transplant.Future Plans: Based on the clinical outcome of the role fucosylated Tregs in preventing GVHD in the double cord blood transplant setting, we plan to extend our clinical trial to include other high risk transplants including mismatched unrelated donor and haplo-identical transplant. In addition, if the preclinical data with fucosyltransferase VII appears to be promising, we will work with the Targazyme company to generate clinical grade FT-VII enzyme for the purpose of a pilot study.

Timeline