Pilot Study of Stem Cell Transplantation for Children and Young Adults With Refractory Crohn's Disease.

Crohn's Disease (CD) is an immunologically mediated chronic illness that has a relapsing and
remitting course, most commonly presenting in the 2nd or 3rd decade and causing lifelong
impairment of health and quality of life. Mainstay of clinical treatment for severe disease
is combination of anti-inflammatory agents like 5-aminosalicyaltes and immunosuppressive
medications like corticosteroids and newer anti-TNF antibodies like Infliximab. None of the
drugs are, at present, curative and a relevant subset of patients are refractory to many of
these pharmacologic approaches.

Immunoablative treatment followed by autologous stem cell rescue (Autologous HSCT) has been
tried in this refractory group of patients with successful results. Autologous HSCT is
effective in this auto-immune setting through the eradication of effector/memory T-cell
clones due to a direct immuno-ablative effect of drugs used in the preparative regimen; by
leading to an immune-reset- recovering clones of T- cells from the infused stem cells do not
mount an auto- immune response and are tolerant to 'self' antigens and, by upregulation of T
regulatory cells (Treg, CD4+CD25+FOXP3+ or CD8+ FOXP3+) via change in cytokine mileu during
transplant. Increased population of Tregs restricts the activity of self-reactive effector
T-cells.

This pilot study is designed to gain on the success of previously published adult studies of
autologous HSCT in refractory CD, with the aim to confirm the feasibility, safety and
efficacy of HSCT and post-transplant cyclophosphamide when given post-HSCT in pediatric
patients and young adults.

Since, this combination of immuno-ablative therapy with post-transplant cyclophosphamide has
not been extensively studied in children with CD, therefore transplant related mortality
(TRM) and severe toxicity (> grade 3 toxicity by NCI criteria) will be monitored for 100
days post-transplant in all the patients and stopping rules will be enforced in case of
excessive toxicity or TRM (>10%). Clinical assessments will be done for 1 year post-HSCT for
disease activity, steroid free remission period to evaluate the 'clinical efficacy' of this
procedure.

The major aim of this pilot is to generate the preliminary safety data to confirm the
feasibility and benefit of autologous HSCT in children and young adults with refractory CD.

Study Design:

This is a single arm, pilot study designed to estimate the feasibility, safety and benefit
of immunoablation with hematopoietic stem cell transplantation (HSCT) using autologous stem
cells followed by administration of post-transplant cyclophosphamide for children and young
adults with refractory Crohn's disease (CD). The study population is patients with severe
and refractory Crohn's disease who are ≥10 and ≤ 30 years of age.

Objectives:

Primary Objective:

To determine the feasibility and toxicity of immunoablation with HSCT and post-HSCT infusion
of cyclophosphamide in children and young adults with refractory Crohn's disease (CD). Death
(transplant related mortality, TRM) and severe non-hematologic toxicity (≥ grade 3 toxicity;
NCI Toxicity Criteria version 4.0) within the first 100 days after HSCT will be monitored to
meet this end-point.

Secondary Objectives:

Secondary objectives are evaluations of incidence of HSCT related complications and
evaluations of response. These include:

1. Incidence of viral reactivations - CMV and EBV

2. Incidence of invasive fungal infections

3. Immune reconstitution after HSCT lymphocyte subpopulations (absolute number of CD3,
CD4, CD8 and CD19 cells), immunoglobulin levels (IgG) and CD4+ CD25+ CD127+ regulatory
T cells (Treg) populations on day +30 , +60 , +100, day +180 and yearly post-HSCT.

4. Evaluate the efficacy and benefit of HSCT in this population at 1 year post HSCT, by
serial assessments of clinical activity of CD, assessment of disease severity- CDAI
scores and length of steroid free remissions.

Hypotheses:

Primary: The HSCT and post-transplant cyclophosphamide regimen will be well tolerated with
acceptable toxicity and will lead to <10% transplant related mortality (TRM).

Background and Rationale:

Crohn's disease (CD) is a chronic illness that is immunologically mediated, of unknown
etiology, but probably induced by an exposure to intestinal bacteria or their component
antigens leading to an excessive T helper type 1 (TH-1) mediated chronic inflammation of the
gastrointestinal (GI) tract 1. Crohn's disease is a relapsing and remitting disorder most
commonly presenting in the 2nd or 3rd decade and causing lifelong impairment of health and
quality of life 2.

Regardless of the therapy used some patients remain seriously ill with active disease after
multiple therapeutic options have been exhausted. In addition, patients with refractory
disease suffer from an inability to eat, frequent nausea, vomiting, diarrhea, malnutrition,
growth retardation, fistulae formation, abdominal pain and psychological distress, ileostomy
or colostomy, and multiple surgeries that may lead to short-gut syndrome, severely affecting
quality of life. Though not well characterized, a distinct excessive mortality exists in
this group of patients 3,4.

Patients with refractory CD have a higher mortality rate compared to normal population
(hazard ratio of 1.73); but deaths are usually related to surgical complications, liver
disease, infections and intestinal cancer in long term survivors 5. Even though the overall
mortality is low in this group of patients, they suffer from significant morbidity,
cortico-steroid side effects and poor quality of life and 50% patients with refractory
disease will need surgical interventions (ileostomy, colostomy) further affecting the
complications rate 2,3.

Many drugs have been used for controlling the symptoms of CD, but no drug is, at present,
curative and a relevant subset of patients are refractory to many pharmacologic approaches
6. Almost all drugs employed in the advanced form of disease, such as immunosuppressives and
immunomodulators, have potential risks. Mainstay of clinical treatment is combination of
anti- inflammatory agents like 5-aminosalicyaltes and immunosuppressive medications like
corticosteroids4. Majority of the patients with severe refractory disease are treated with
anti- Tumor Necrosis Factor (TNF) antibodies like Infliximab (Remicade), but limited options
exist for patients who have failed to respond to anti-TNF therapy7. Approximately 40% of
patients with CD develop intolerable side effects or fail to respond to anti-TNF therapy,
hence there is a significant population of this subset of patients where options are very
limited 8.

Some symptoms may be controlled by prolonged steroids, but there are many known long term
effects of continued steroid use- muscle loss, osteopenia, cataracts and cushingoid habitus.
Majority of these patients enroll on other experimental Phase I/II drug trials or resort to
surgical intervention to control symptoms. Surgical approach (partial gut resection of the
inflamed segment with ileo-colostomy) is an option, but is associated with infection risk,
psycho-social and cosmetic issues and increased morbidity due to short gut syndrome,
especially in young adults. Increased mortality is also associated with surgical
interventions 2,3.

Therefore, any intervention that avoids surgery or long term corticosteroids in young adults
may be beneficial. Given that hematopoietic stem cell transplantation (HSCT) have been of
some value in other auto-immune diseases characterized by loss of immune tolerance and or a
TH-1 predominant immune response, it is possible that these procedures could be of value in
CD 9,10. Autologous transplantation might also be of benefit because clearing the body of
committed lymphocyte clones might restore the patient to the status quo ante of being
predisposed to CD but not suffering from it 11.

Rationale of HSCT for CD

HSCT may provide benefit in auto-immune disease by:

1. Eradication of effector/memory T-cell clones due to a direct immuno-ablative effect of
drugs used in the preparative regimen.

2. Immune-reset: recovering clones of T-cells from infused stem cells do not mount an
auto-immune response and are tolerant to 'self' antigens.

3. Upregulation of T regulatory cells (Treg, CD4+CD25+FOXP3+ or CD8+ FOXP3+) - by change
in cytokine mileu during transplant. Increased population of Tregs restricts the
activity of self-reactive effector T-cells.

4. Engraftment of uncommitted stem cells in the gut mucosa may lead to direct healing of
mucosa.

Evidence for the effectiveness of HSCT in Crohn's disease Initial observations were made in
patients with Crohn's disease and hematologic malignancies. For patients who underwent HSCT
for their hematologic malignancy they had remission of their Crohn's disease 12.

Several studies have reported the clinical course of patients with Crohn's disease receiving
autologous transplantation for another condition. Perhaps the most interesting concerned a
patient who developed symptoms at the age of 9, who was diagnosed with Crohn's disease 4
years later and who required substantial treatment over the next 7 years 13. Following
autologous stem cell transplantation for non-Hodgkin's lymphoma it was reported that there
was no clinical or laboratory evidence of recurrence of his Crohn's disease in the next 7
years. Another reported case of Crohn's disease had diffuse pan colitis before
transplantation and was asymptomatic (but had inflammation) at endoscopy 3 years later 14.
Another patient who had symptomatic Crohn's disease for 2 years and had undergone surgery
was free of disease 5 years after transplantation. Systematic colonoscopic evaluation showed
persisting subclinical inflammation initially with progressive clearance. At Vanderbilt we
performed an unrelated donor transplant for a patient with AML and Crohn's disease. He is
currently 10 years post-transplant with no symptoms of Crohn's disease.

HSCT specifically for Crohn's disease:

The first report of stem cell transplantation for Crohn's disease concerned 2 patients from
the Chicago group 15. In both of the initial cases Crohn's disease activity index was more
than 250 (normal range <150, pathological range 220 - 600) despite treatment with
infliximab. In both patients, peripheral blood stem cells (PBSC) were mobilized using
cyclophosphamide and granulocyte colony stimulating factor (G-CSF), enriched ex-vivo by CD
34+ selection. Cyclophosphamide and antithyomcyte globulin (ATG) were used for immune
conditioning prior to transplantation. The first patient was a 22 year old woman with a past
history of a right hemicolectomy with severe ileocolic Crohn's disease causing intractable
diarrhea with 25 bowel actions per day, fistula and peri-anal sepsis. The second was a 16
year old boy who had been unwell for 6 years requiring tube feeding and receiving
methotrexate, 6-mercaptopurine and 5 amino acyclic acid with Crohn's colitis. In both of
these cases, following transplantation, diarrhea resolved and the Crohn's disease activity
index normalized. In the first patient the haemoglobin rose, whereas in the second it
remained at a slightly sub-normal level. Post-transplantation CRP was within normal limits
and the albumin remained or became normal. These cases were accompanied by extensive
colonoscopic evidence of disease activity. Prior to transplantation both cases had areas of
severely active Crohn's disease with cobble stoning, fissuring and deep ulceration.
Following transplantation the bowel remained abnormal though changes were much more trivial
with superficial erosions only.

Oyama et al reported on autologous HSCT for 12 patients median age of 27 (15-38) with severe
refractory Crohn's disease 16. The conditioning regimen consisted of cyclophosphamide (200
mg/kg) and equine ATG (90 mg/kg). Eleven of 12 patients entered a sustained remission
defined by a CDAI < 150. After a median follow-up of 18.5 months (range, 7-37 months), only
one patient has developed a recurrence of active CD, which occurred 15 months after HSCT.

Hasselbatt et al reported on autologous HSCT 12 patients (ages 24-50) with refractory
Crohn's disease using high dose cyclophosphamide (200 mg/kg) without ATG 17. PBSC harvest
following mobilization chemotherapy was successful in 11/12 patients and resulted in a
clinical and endoscopic improvement in 7/12 patients. Subsequent conditioning and autoPBSCT
were performed in nine patients and were relatively well tolerated. Among those, five
patients achieved a clinical and endoscopic remission within 6 months after autoPBSCT.
However, relapses occurred in 7/9 patients during follow-up, but disease activity could be
controlled by low-dose corticosteroids and conventional immunosuppressive therapy.

In 2010 Burt et al reports on autologous HSCT in 24 patients with mean age of 24 years
(range 15-52) with anti-TNF refractory Crohn's disease 18. Stem cells were mobilized from
the peripheral blood using cyclophosphamide (2.0 g/m2) and G-CSF (10 ug/kg/day), enriched ex
vivo by CD34 selection, and reinfused after immune suppressive conditioning with
cyclophosphamide (200 mg/kg) and either equine antithymocyte globulin (ATG, 90 mg/kg) or
rabbit ATG (6 mg/kg). Eighteen of 24 patients are 5 or more years after transplantation. All
patients went into remission with a CDAI less than 150. The percentage of clinical
relapse-free survival defined as the percent free of restarting CD medical therapy after
transplantation is 91% at 1 year, 63% at 2 years, 57% at 3 years, 39% at 4 years, and 19% at
5 years.

The percentage of patients in remission (CDAI < 150), steroid-free, or medication-free at
any posttransplantation evaluation interval more than 5 years after transplantation has
remained at or greater than 70%, 80%, and 6% respectively.

Regulatory T cells (Tregs) and Auto-immune disease:

Regulatory T cells (Tregs) are implicated in the suppression of immune responses and in
maintenance of tolerance 19,20. The percentage of CD4+CD25+FOXP3+ T cells (T regs) is
significantly decreased in patients with auto-immune diseases like SLE and Type 1 diabetes
20. Also, transfer of Treg can prevent auto-immune phenotype that develops after Treg
depletion in animal models.

During HSCT for auto-immune diseases, an increase in Treg population correlates with
remission induction. Zhang et.al has shown that HSCT leads to a newly generated population
of Treg cells that maintain patients in true immunological remission 21.

Burt et.al, also evaluated the role of Treg cells in some of their patients who underwent
HSCT for CD. Before HSCT the mean % of Treg cells was significantly low in CD patients
(2.62% ± 2.01%), compared to normal volunteers (3.86% ± 1.67%). A robust increase in the %
of Treg was observed after HSCT, but this was not sustained over period of time and may have
contributed to relapses 18. Hence, there is clinical evidence that a sustained increase in
Treg population may maintain remission in patients with auto-immune disorder.

Rationale of the proposed Study:

This pilot study is designed to estimate the feasibility of HSCT and safety of post-HSCT
cyclophosphamide infusions for patients with refractory disease who have failed to respond
to Infliximab/and or other immunosuppressive agents. This is designed as a small pilot study
with built in 'stopping rules' in case of excessive toxicity or death.

Rationale for the immuno-ablative regimen:

Rationale of CY for mobilization: Cyclophosphamide (CY) will be used for mobilization of
peripheral stem cells and for immuno-ablation prior to HSCT. CY has been traditionally used
for mobilization in all HSCT for refractory CD.

Rationale for post-transplant Cyclophosphamide (CY)- Recently, use of post-transplant CY has
been pioneered by John Hopkins and shown to be a very effective GVHD prophylaxis in patients
undergoing HSCT for hematologic malignancies, even in the setting of HLA mismatched and
haploidentical donors. Post-transplant delivery of CY allows for effective deletion of
proliferating allo-reactive T- cells that cause GVHD without adding to the toxicity of the
regimens 22. In a recent study by the Hopkins group post-transplant cyclophosphamide was
successfully used in patients with sickle cell disease undergoing haploidentical bone marrow
transplant with no toxicities.

Some studies have performed CD34+ stem cell selections to get rid of all immune-reactive T-
cells clones and use purified CD34+ infusions for regenerating the immune system 18. The
problem with this approach is delayed immune-reconstitution post-transplant and a higher
incidence of infections.

European trials have used un-manipulated peripheral blood stem cells (PBSCs) as infusions
(without any T cell depletion or CD34+ selections) for SCT in auto-immune diseases 23.
Since, peripheral stem cells contain the same T cells clones that are immuno-reactive and
are infused along with the donor stem cells in non-T cell depleted infusion, this may be one
of the reasons for relapse in T cell replete HSCT done till date for CD.

Therefore, to overcome the above-mentioned issues, we propose to use un-manipulated PBSCs,
but give two doses of CY pre- and two doses post-stem cell infusion to effectively deplete
the immuno-reactive T cell clones present in the autologous infusion and therefore, decrease
the incidence of relapse. The total dose of CY remains the same (200 mg/kg), therefore, it
will not add to the toxicity of the regimen, but may lead to more effective depletion of the
immune- reactive clones, obviating the need for costly in-vitro T cell depletions/CD34+
selections.

Also, CY causes sustained depletion of all T cell subsets, but more profound depletion of
CD4+ CD25- subsets, resulting in relative enrichment of CD4+CD25+ cells, which we hope to
expand by using post-transplant cyclophosphamide.

Rationale for Rabbit-ATG (Thymoglobulin, Genzyme Corp, Cambridge, MA): will also be used for
effective immune-ablation and for in-vivo T cell depletion post- stem cell infusion. Rabbit
ATG will be used as this has been shown to promote the expansion of functional Tregs
in-vitro 24. Rabbit ATG was use in the largest study by Burt et al 18.

(Please note reference numbers can be found on pages 25-26 of the protocol.)

Inclusion Criteria:

All the subjects considered eligible for the study will be screened and reviewed by the
Gastroenterology (GI) physicians prior to enrollment (Dedrick Moulton, MD or his designee
at Vanderbilt Children's Hospital) 2. Age ≥ 10 and < 30 years 3. Disease status:

1. Confirmed diagnosis of Crohn's Disease: Diagnosis of Crohn's disease that has been
established based on typical endoscopic/histologic and/or radiological appearances.

2. Active disease, defined as: Pediatric Crohn's Disease Activity Index (PCDAI) >30 (see
Appendix I) or Crohn's Disease Activity Index (CDAI) of >250 (see Appendix II) at any
time within 3 months prior to enrollment and any one of the following- i. Endoscopic
evidence of active disease confirmed on histology within 3 months prior to
enrollment, or ii. Clear evidence of active small bowel Crohn's disease on small
bowel imaging within 3 months prior to enrollment.

3. Refractory disease- Moderate to severe disease that has been unresponsive to current
or prior therapy with mercaptopurine and/or azathioprine (thiopurines), methotrexate
and anti-TNF therapy. Patients should have relapsing disease (i.e. ≥ 1
exacerbation/year) or corticosteroid dependence despite current or prior thiopurines,
methotrexate and anti-TNF maintenance therapy or clear demonstration of intolerance
or toxicity to these drugs. Patients who fail induction therapy with corticosteroids
and anti-TNF therapy, and are therefore not eligible to receive maintenance therapy
with thiopurines or methotrexate will also be candidates for enrollment.

4. Negative stool culture, C. difficile, and negative CMV pcr (in stool or colonic
biopsy). Patients with CMV colitis will receive a trial of anti-viral treatment and
only responders will be considered eligible for inclusion.

5. Patients with a prior ileostomy or colostomy may enter the study. For this group
of patients', physician's global assessment will be used to assess clinical activity
of CD, as Pediatric CDAI and CDAI scoring method may not be representative of disease
activity.

6. Patients with abscesses are eligible to enroll once the abscesses or any other
significant infection has resolved.

Exclusion Criteria:

1. Pregnancy or unwillingness to use adequate contraception during the study- if a woman
is of childbearing age.

2. HIV infection.

3. Organ function criteria-

1. Renal: creatinine clearance < 50 ml/min/1.73m2 (measured or estimated).

2. Cardiac: left ventricular ejection fraction <30% by multigated radionuclide
angiography (MUGA) or a shortening fraction of < 25% by cardiac echocardiogram.

3. Pulmonary Function tests: DLCO < 30% or patient on oxygen.

4. Hepatic: serum bilirubin > 3 mg%; AST and ALT > 3x ULN for the institutional
lab.

4. Uncontrolled Hypertension (using age based criteria) despite at least 2 anti-
hypertensive agents.

5. Active Infection or risk thereof-

1. Current abscess or significant active infection

2. Perianal infection is not an exclusion criterion, provided there is drainage
with or without placement of drain.

3. Abnormal chest x- ray (CXR) consistent with active infection or neoplasm.

6. Severe diarrhea due to short small bowel; patients believed to have < 700 mm of small
bowel and diarrhea attributable to this will be excluded.

7. Patients with toxic megacolon, active bowel obstruction or intestinal perforation.

8. Unable to collect minimum of 3 x106/kg CD34+ cell dose. These patients will be
excluded from receiving the preparative regimen.