Clinical trial NCT02200445
Conditions: Ulcerative Colitis
Keywords: Ulcerative colitis Inflammatory bowel disease Interleukin 2 Interleukin-2 IL-2 Regulatory T Cells Tregs T-Lymphocytes, Regulatory
Sponsor: Scott B. Snapper, MD PHD
Start date: 2015-02-01; Completion date: 2021-03-01
Enrollment: 26 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to determine the safety and maximum effective dose (MED) of Interleukin-2 in subjects with moderate-to-severe ulcerative colitis.

Detailed description:
Interleukin-2 (IL-2) is a T cell growth factor. IL-2 is currently licensed for the treatment of metastatic renal cell carcinoma and metastatic melanoma, where it promotes the expansion of anti-cancer cytotoxic T cells and natural killer (NK) cells. However at low doses (100-times lower than those used in cancer therapy), IL-2 promotes the selective expansion of regulatory T cells (Tregs): an immune modulating subset of CD4+ lymphocytes.

A recent phase 1 clinical trial from our collaborators at the Dana Farber Cancer Institute showed that low-dose IL-2 selectively expands Tregs in patients with treatment-resistant Graft vs. Host Disease (GvHD), and that low-dose IL-2 is safe in this condition. A detailed immunological analysis of samples from this study showed that low-dose IL-2 treatment was associated with increased Treg proliferation, increased de novo thymic generation of Tregs, and a resolution of defects in intracellular signalling and apoptosis seen in Tregs in chronic GvHD. A recent phase 1 study from another group showed that low-dose IL-2 is safe in the treatment of HCV-associated vasculitis. Low-dose IL-2 has also been shown to be well-tolerated in subjects with HIV.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon. Evidence from pre-clinical models of intestinal inflammation, and also from patients with monogenetic defects in Treg function, suggests that Tregs play a role in the prevention of inflammation in the intestine.

The treatment (or intervention) in this study is a once-daily, subcutaneous injection of IL-2, for a total of 8 weeks. The first 2 doses of the study drug will be administered by research nurses at Boston Children's Hospital. Further doses will be self-administered, at home. Training will be provided for correct self-administration.

This is a 3+3 dose escalation study of IL-2 in moderate-to-severe UC. This study design is powered to identify the MED of low-dose IL-2 in UC. Once the MED is identified, a further 10 subjects will receive IL-2 at that dose. Recruitment of between 2 and 28 patients is planned. The maximum tolerated dose (MED) is the highest tolerated dose level at which a minimum of 6 subjects have been evaluated, with fewer than 2 evaluable subjects in 6 experiencing a dose limiting toxicity (DLT); i.e. DLT in \>1/6 evaluable subjects. In addition to the above at least 1 patient should meet the criteria for response or remission for it to be considered the MED.

Dose levels are based on the experience of our collaborators in GvHD. In addition to determining the MED, this study will determine if low-dose IL-2 is safe and well-tolerated in patients with moderate-to-severe ulcerative colitis. A detailed immunological analysis of samples obtained from this study will determine if low-dose IL-2 expands Tregs in vivo, in patients with moderate-to-severe UC. Immunological changes will then be correlated with clinical response.

The study will take place at Boston Children's Hospital. The study will involve 10 study visits. Most of the study visits involve blood tests. A flexible sigmoidoscopy or colonoscopy will be performed as part of the screening process. A flexible sigmoidoscopy will also be performed on completion of therapy, to determine clinical response.

The first two subjects to receive the study drug will be admitted overnight following the first dose. Subsequent doses will be administered on an out-patient basis. All other subjects will receive IL-2 on an out-patient basis.

Responders (with an acceptable side-effect profile) will be allowed to continue the study drug for at least 1 year. Compensation will be provided for participants.

Arms: arm 1: Study drug: Interleukin-2 (aldesleukin, Proleukin, IL-2).

Each subject will receive an 8-week course of once-daily, subcutaneously administered IL-2. There will be three dose cohorts. Each subject will be recruited into a single dose cohort and receive a single dose level of IL-2 throughout the study.

The dose levels will be as follows:

* Cohort 1: 0.3x10\^6 IU/m\^2/day.
* Cohort 2: 1.0x10\^6 IU/m\^2/day.
* Cohort 3: 1.5x10\^6 IU/m\^2/day.

Up to 6 subjects will be recruited to each dose cohort.

Once the maximum effective dose has been identified, a further 10 subjects will receive IL-2 at the maximum effective dose.
Interventions: intervention 1: Interleukin-2 (aldesleukin).
Intervention descriptions: intervention 1: Description of intervention is covered in "Arm", above.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
Page 1 of 56 
Clinical Trial Protocol 
LOW DOSE IL-2 FOR THE TREATMENT OF ULCERATIVE COLITIS 
 
Clinical Investigators: 
Scott Snapper, MD PhD 
Joshua Korzenik MD 
 
James Canavan MD PhD 
Athos Bousvaros MD 
 
Jodie Ouahed MD 
Jessica Allegretti MD 
 
Matthew Hamilton MD 
 
 
 
Laboratory Investigators: 
Vanessa Mitsialis, MD 
 
Naresh Redhu PhD 
 
 
Research Coordinators 
Jonathan Hurtado 
Caroline Rourke 
Noah Herwood 
 
Julia Green 
Sydney Whitcomb 
Study Statistician: 
Enju Liu PhD 
 
Collaborators 
John Koreth MB BS DPhil 
Robert Soiffer MD  
Jerome Ritz MD 
 
Protocol Version (Date): 
Version 6 (May 11,2016) 
FDA IND reference: 
122161. Submitted 3/17/2014. Study May Proceed : 4/15/2014. 
IRB reference: 
Boston Children’s Hospital IRB-P00009599.  
Submitted 4/22/2014.  Approved 9/15/2014. 
ClinicalTrials.gov identifier: 
NCT02200445 
 


 
 
 
Page 2 of 56 
1 
TABLE OF CONTENTS 
1 
Table of Contents .................................................................................................................. 2 
2 
List of Tables ........................................................................................................................ 6 
3 
Protocol Synopsis. ................................................................................................................ 7 
3.1 
Study Title. ............................................................................................................. 7 
3.2 
Objective. ............................................................................................................... 7 
3.3 
Subject Eligibility..................................................................................................... 7 
3.4 
Registration Contacts. ............................................................................................ 7 
3.5 
Schema. ................................................................................................................. 7 
3.6 
Evaluation. ............................................................................................................. 7 
4 
Background and Significance. ............................................................................................... 8 
4.1 
The Current Requirement for Novel Therapies in Ulcerative Colitis. ....................... 8 
4.2 
Regulatory T Cells and Mucosal Inflammation in UC. ............................................. 9 
4.3 
The Rationale for High Dose IL-2 as a Cancer Therapy: Induction of Effector 
Immune Cells to Enhance Anti-Tumor Immunity. ............................................... 10 
4.4 
The Rationale for Low Dose IL-2 in UC: Selective Induction of Regulatory T 
Cells to Modulate Immune Responses. .............................................................. 11 
4.5 
Preclinical and Clinical Studies of Low Dose IL-2 to Selectively Expand 
Regulatory T Cells In Vivo in Man. ..................................................................... 12 
4.6 
Safety Profile of Low Dose IL-2:  Side-Effects Seen in Recent Clinical Trials. ...... 13 
4.7 
Hypothesis............................................................................................................ 14 
5 
Study Design and Objectives. ............................................................................................. 15 


 
 
 
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5.1 
Study Design. ....................................................................................................... 15 
5.2 
Primary Objectives. .............................................................................................. 15 
5.3 
Secondary Objectives. .......................................................................................... 15 
6 
Subject Selection. ............................................................................................................... 16 
6.1 
Inclusion Criteria. .................................................................................................. 16 
6.2 
Exclusion Criteria.................................................................................................. 17 
7 
Rules for Concomitant IBD Medications. ............................................................................. 19 
8 
Subject Entry. ..................................................................................................................... 20 
8.1 
Recruitment and Accrual. ..................................................................................... 20 
8.2 
Study Site Responsible Investigator. .................................................................... 20 
9 
Treatment Program. ............................................................................................................ 21 
9.1 
Description of Study Design. ................................................................................ 21 
9.2 
Dose Cohorts. ...................................................................................................... 21 
9.3 
Dose Escalation Schema. ..................................................................................... 22 
9.3.1 
Dose Level A. ......................................................................................... 22 
9.3.2 
Dose Level B. ......................................................................................... 23 
9.3.3 
Dose Level C. ........................................................................................ 24 
9.3.4 
Recruitment of Additional Subjects. ........................................................ 24 
9.4 
Determination of Maximum Tolerated Dose. ......................................................... 25 
9.5 
Definition of Dose Limiting Toxicities. ................................................................... 26 
9.6 
Clinical Assessments for Toxicity. ......................................................................... 27 


 
 
 
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9.7 
Extended Treatment with Low-Dose IL-2. ............................................................. 27 
10 Study Drug: Aldesleukin. ..................................................................................................... 28 
10.1 
Formulation, Reconstitution and Storage. ........................................................... 28 
10.2 
Reconstitution. .................................................................................................... 28 
10.3 
Storage. .............................................................................................................. 29 
10.4 
Dosage. .............................................................................................................. 29 
10.5 
Administration. .................................................................................................... 30 
11 Clinical and Laboratory Evaluations. ................................................................................... 31 
11.1 
Screening (Pre-Treatment) Evaluation (Week -2). .............................................. 31 
11.2 
Baseline, Treatment and End-of-Study Evaluations (Day 0 to Week 6 and 
Week 12). .......................................................................................................... 32 
11.3 
End of Treatment Evaluation (Week 8). .............................................................. 33 
11.4 
Evaluation of Subjects Undergoing Extended Treatment with Low Dose IL-2. .... 34 
11.5 
Details of Specific Assays. .................................................................................. 35 
11.6 
Assessment of Aldesleukin Immunogenicity. ...................................................... 35 
11.7 
Anticipated Toxicities. ......................................................................................... 38 
11.8 
Criteria for Treatment Discontinuation. ............................................................... 39 
12 Reporting of Unanticipated Problems Involving Risks To Research Subjects, Including 
Adverse Events. ........................................................................................................................ 40 
12.1 
Reporting to the Principal Investigator. ............................................................... 40 
12.2 
Reporting to the Institutional Review Board. ....................................................... 41 
12.3 
Reporting to Prometheus Laboratories. .............................................................. 42 


 
 
 
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13 Data Safety And Monitoring Board. ..................................................................................... 42 
14 Study Endpoints. ................................................................................................................. 43 
14.1 
Determination of Safety of Daily SC Low Dose IL-2. ........................................... 43 
14.2 
Determination of the Maximum Tolerated Dose of Daily SC Low Dose IL-2. ....... 43 
14.3 
Determination of the Feasibility of Administering an Eight Week Course of 
Daily SC Low Dose IL-2 in Subjects with Moderate to Severe UC. .................... 43 
14.4 
Determination of the Clinical Response to Daily SC Low Dose IL-2. ................... 43 
14.4.1 Definition of Clinical Response. .............................................................. 43 
14.4.2 Definition of Remission.. ......................................................................... 44 
14.4.3 Definition of Mucosal Healing. ................................................................ 44 
14.5 
Assessment of the Immunological Response to IL-2 Therapy. ............................ 44 
15 Statistical Considerations. ................................................................................................... 46 
15.1 
Probability of Dose Escalation Under Various DLT Rates. .................................. 46 
15.2 
Accrual. .............................................................................................................. 46 
16 Ethics and Good Clinical Practice. ...................................................................................... 47 
17 References. ........................................................................................................................ 48 
18 Appendix A:  Mayo Scoring Worksheet. .............................................................................. 53 
19 Appendix B:  Common Terminology Criteria for Adverse Events. ........................................ 56 
 
 


 
 
 
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2 
LIST OF TABLES 
Table 1: Dose Escalation Cohorts ............................................................................................. 22 
Table 2: Rules for Dose Escalation and Allowable DLTs Per Cohort ......................................... 25 
Table 3: Summary of Required Data ......................................................................................... 37 
Table 4:  Anticipated Toxicities ................................................................................................. 38 
Table 5:  Mayo Scoring System for Assessment of Ulcerative Colitis Activity ............................ 44 
Table 6: Probability of Dose Escalation ..................................................................................... 46 
 
 
 


 
 
 
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3 
PROTOCOL SYNOPSIS. 
3.1 
Study Title. 
 
A Phase I Study of Low Dose Subcutaneous Interleukin-2 (IL-2) For The Treatment of 
Ulcerative Colitis. 
3.2 
Objective. 
 
To determine the safety and maximum tolerated dose of daily subcutaneous IL-2 in the 
treatment of moderate-to-severe ulcerative colitis (UC). 
3.3 
Subject Eligibility. 
• 
Adult subjects with moderate-to-severe UC (Mayo score 6-12). 
• 
Declined, or failed to tolerate or respond to a standard course of UC medication 
prescribed with the intention of inducing or maintaining remission. 
3.4 
Registration Contacts. 
• 
Scott Snapper MD PhD (Boston Children’s Hospital):  
(617) 919-4973 
• 
James Canavan MD (Boston Children’s Hospital):  
(617) 642-1841 
3.5 
Schema. 
Assessments 
 
 
 
  
 
 
  
 
 
 
 
 
 
 
 
Screening 
 
Treatment 
 
 
 
 
Week 
-2 
-1 
0 
      1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
3.6 
Evaluation. 
• 
Assessment of study drug toxicity. 
• 
Assessment of UC disease activity and mucosal healing. 
 


 
 
 
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4 
BACKGROUND AND SIGNIFICANCE. 
4.1 
The Current Requirement for Novel Therapies in Ulcerative Colitis. 
Ulcerative Colitis (UC) is a chronic inflammatory disease of the rectum and colon, causing 
bloody diarrhea, urgency and tenesmus1.  UC has a population-based incidence of 14/100,000, 
with a prevalence of 240/100,000, affecting an estimated 750,000 people in the United States2,3.  
Mucosal inflammation in UC is managed with anti-inflammatory and immune-modulating drugs 
to induce remission (e.g. mesalamine, corticosteroids, biologic agents) and, in patients with 
frequent flares in disease activity, immune-modulating drugs to maintain remission (e.g. 
azathioprine, 6-mercaptopurine [6-MP], methotrexate, biologic agents).  A substantial proportion 
of patients have sub-optimal responses to current therapies or develop adverse events.  
Furthermore, surgical resection of the colon and rectum is the only currently available “curative” 
treatment for those with refractory disease.  However, surgery is unpopular amongst patients 
and is associated with its own spectrum of complications. Consequently, an urgent need exists 
to identify new, more effective therapies for UC with more acceptable safety profiles. 
 
A significant unmet need persists for new approaches to therapy as existing medications 
have limited benefit in UC.  The results of clinical trials to induce remission in UC are surprisingly 
modest.  In mild-to-moderate UC, mesalamine induces remission in approximately 40% of 
patients4.  As much as a third of newly-diagnosed UC patients will have a need for oral or 
parenteral corticosteroids to induce remission5.  In these patients, corticosteroids induce 
remission in over half, with a partial response in an additional third, but at one year 28% of those 
initially treated with steroids are steroid-dependent and a third require surgery5.  The key finding 
of Truelove & Witts’ 1955 study of cortisone in UC was that steroids reduced mortality in this 
previously fatal disease6.  In patients with moderate-to-severe UC, 69% respond to infliximab, 
with half of those entering remission, but 31% fail to respond7.  Many who initially respond to the 
anti-TNF medications now indicated for UC (infliximab, adalimumab or golimumab) will loose 
response over time.  Longer term use of immune modulating drugs to maintain remission is also 
not without risks, with clinical effectiveness always being balanced against untoward side 
effects, including anemia, pancreatitis, lymphoproliferative disease and infection8,9.  Finally, a 
number of novel agents targeting aspects of the mucosal immune system, such as vitulizumab10 
(an anti-CD3 monoclonal antibody), or secukinumab11 (an anti-IL-17A monoclonal antibody) 
have not yielded promising results in recent clinical trials in IBD.   


 
 
 
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Colectomy rates reflect a failure of medical management of UC and an unmet therapeutic 
need.  Six percent undergo colectomy within a year of diagnosis12.  In patients with moderate to 
severe colitis, requiring corticosteroid treatment at presentation, this figure rises to 29%5.  
Overall, 24% will have had a colectomy by the 10th year of disease13.  Despite a variety of new 
medications, the rate of colectomy in UC has not substantially changed in the last five 
decades14,15, indicating that current medicines have failed to alter the natural history of UC. 
4.2 
Regulatory T Cells and Mucosal Inflammation in UC. 
 
Gut mucosal homeostasis is characterized by a constant interaction between commensal 
intestinal microbiota and the mucosal immune system.  In order to prevent inappropriate 
inflammation to commensal bacteria in the gut, a population of thymus-derived regulatory T cells 
(termed “natural” Tregs) characterized by CD4+CD25+FOXP3+ expression in mice and 
CD4+CD25hiCD127loFOXP3+ expression in humans allows tolerance to “self-antigens”16.  
Activation of Tregs by inflammatory stimuli causes them to antagonize the activation and effector 
function of a range of pro-inflammatory immune cells that are contemporaneously activated by 
similar stimuli.  In humans, this includes reduced proliferation and cytokine production by 
responder CD4+, CD8+ and  T cells, and modulation of antigen presenting cells (APC) and 
natural killer (NK) cell responses17-20.  Furthermore, Tregs are effective at treating murine 
intestinal inflammation in vivo.  By antagonizing activation of conventional effector CD4+CD25lo-int 
T cells (Tcon), Tregs both prevent the onset of colitis21,22 and treat established colitis23 in the Tcon 
mediated CD4+CD45RBhi adoptive transfer model of colitis in mice.  Tregs also treat colitis 
mediated by the innate immune system in both the RAG2-/- 129/SvEv Helicobacter hepaticus24 
and TRUC (T-bet-/- x RAG2-/- UC)25 mouse models of colitis. 
 
The consequences of defective Tregs can be best appreciated in the X-linked Foxp3 
mutant scurfy mouse, where hemizygous males develop lethal multi-organ autoimmunity, 
including enteritis, due to CD4+ hyperactivation26.  In humans, FOXP3 mutations are associated 
with the IPEX syndrome (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked), 
which causes gastrointestinal inflammation due to dysregulated lymphocyte proliferation27.   
 
Treg defects are also seen in Wiskott-Aldrich syndrome, an X-linked primary human 
immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and 
autoimmunity28.  Our laboratory and others identified that defective expression of Wiskott-Aldrich 
syndrome protein (WASP) results in decreased Treg numbers in vivo and decreased proliferation 


 
 
 
Page 10 of 56 
and expression of IL-10 and TGF in vitro28-30.  WASP-/- Tregs are also unable to prevent 
CD45RBhi colitis in mice 28.  Interestingly, WASP deficiency in the innate immune system also 
impairs the ability of adoptively transferred wild type (WT) Tregs to suppress naïve T cell-
mediated colitis, although this can be overcome by increased Treg dose31.   
 
WASP-/- Tregs are defective in IL-10 secretion28.  We also recently identified causative 
interleukin-10 receptor (IL-10R) polymorphisms responsible for the development of infantile IBD 
that, at least in part, may be associated with defective Treg function32,33.  In this regard, ablation 
of IL-10R in murine Tregs impairs STAT3-dependent suppression of IL-17-mediated colitis34.  
These observations support a role for Tregs in preventing inappropriate intestinal inflammation in 
humans. 
 
In humans in health, CD4+CD25hiCD127loFOXP3+ Tregs comprise 5-10% of peripheral 
blood (PB) CD4+ lymphocytes and 10-15% of lamina propria (LP) CD4+ lymphocytes.  The PB 
Treg fraction is decreased in acute (diverticulitis) and chronic (UC) mucosal inflammation, while 
colonic LP Tregs are increased in active IBD35,36.  The frequency of PB and LP Tregs normalizes in 
both quiescent and treated UC35,36, consistent with the hypothesis that Tregs migrate and/or are 
induced de novo in the setting of inflammation. 
4.3 
The Rationale for High Dose IL-2 as a Cancer Therapy: Induction of Effector 
Immune Cells to Enhance Anti-Tumor Immunity. 
 
IL-2 is a four-bundle -helical cytokine secreted by many cell types but is primarily 
secreted by CD4+ lymphocytes in secondary lymphoid organs under steady-state conditions and 
is strongly induced on antigen-specific activation37.  At high concentrations, IL-2 activates 
multiple cell types, inducing Tcon proliferation and Th1 polarization via STAT5-dependent 
expression of IL-12R2 and T-bet38, Th2 polarization via STAT5-dependent early expression of 
IL-4R and chromatin remodelling at the Il-4 locus39.  It is also responsible for NK cell activation 
and proliferation37, and helping primary and secondary expansion of CD8+ T cells37. 
 
Intravenous (IV) and subcutaneous (SC) IL-2 is currently licensed for the treatment of 
metastatic melanoma and metastatic renal cell carcinoma.  At the high doses of IL-2 approved 
for the management of these malignancies, IL-2 induces Tcons, cytotoxic CD8+ T cells and NK 
cells37, with the intention of boosting anti-cancer immunity.  Approximately 15% of patients 


 
 
 
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develop a clinical response40.  IL-2-mediated immune activation also leads to secretion of TNF, 
IFN and IL-15, which is associated with side-effects40.   
 
Common side-effects of high dose IL-2 therapy in the treatment for metastatic melanoma 
or metastatic RCC include: flu-like symptoms, diarrhea, eosinophilia, capillary leak syndrome, 
impairment of neutrophil function and risk of Gram positive infection and sepsis40.  Early studies 
reported treatment-related mortality of 4%, primarily related to a sepsis-like syndrome40.  
Kammula and colleagues treated 1241 patients with IL-2 at the National Cancer Institute 
between 1985 and 199741.  They noted a significant decrease in Grade 3/4 toxicity over time.  
Comparing the first and last cohorts of 155 patients, serious diarrhea decreased from 92% to 
12%, hypotension from 81% to 31%, neuropsychiatric toxicity from 19% to 8%, line sepsis from 
18% to 4%, tracheal intubations from 12% to 3%, and cardiac ischemia from 3% to 0%41.  
Treatment-related deaths were not seen in the final 809 patients.  The authors attributed the 
improvement in safety profile to judicious pre-treatment screening, patient optimization and 
improved recognition and management of treatment-related toxicities. 
4.4 
The Rationale for Low Dose IL-2 in UC: Selective Induction of Regulatory T Cells to 
Modulate Immune Responses. 
 
IL-2 signals through a dimeric receptor composed of CD122 (IL-2R) and the c chain (IL-
2R)42.  CD4+CD25hi Tregs constitutively express CD25 (IL-2R), a non-signaling molecule that 
readily associates with the dimeric IL-2 receptor to form a trimeric receptor complex.  The 
trimeric IL-2R, involving CD25, has a 10-100-fold higher affinity for IL-2, compared to the dimeric 
IL-2R, without CD2537,43.  In contrast, CD4+CD25lo-int Tcons constitutively express the dimeric IL-
2R and only transiently express CD25 on activation42.  This confers distinct IL-2 response 
profiles to CD4+CD25hi Tregs and CD4+CD25lo-int Tcons in man.  Our collaborators have shown that 
in vitro treatment of human PB CD4+ lymphocytes with low-dose IL-2 (1-10 IU/ml) results in 
STAT5 phosphorylation and proliferation in Tregs but not Tcons43.  This differential response is lost 
in vitro at higher IL-2 concentrations (>100 IU/ml)43,44, consistent with observations that IL-2 
expands both Tregs and Tcons in vivo in man, at the doses used for anti-cancer therapy44,45.  This 
may explain the limitation of IL-2 in cancer therapy, as Treg generation may limit anti-cancer 
immunity.  
 
Constitutive expression of CD25 is important for Treg biology, as IL-2 has a non-
redundant function in promoting peripheral tolerance in vivo46,47.  Genetic absence of 


 
 
 
Page 12 of 56 
components of the IL-2 pathway, such as Il2, Il2ra, Il2rb or Stat5a, or neutralization of IL-2 
results in impaired Treg development, survival and function, leading to lymphoproliferation and 
autoimmunity in mouse models37.    
4.5 
Preclinical and Clinical Studies of Low Dose IL-2 to Selectively Expand Regulatory 
T Cells In Vivo in Man. 
 
Because Tregs respond to much lower doses of IL-2 than pro-inflammatory immune cells, 
“low-dose” IL-2 (some 100-fold lower than that used in anti-cancer therapy) is a conceptually 
attractive approach to selectively expanding Tregs in order to enhance their immune modulating 
effects in diseases characterized by unopposed immune activation. 
 
In mouse models, low-dose IL-2 prevents graft vs. host disease (GvHD) following HLA-
mismatched allogeneic bone marrow transplantation (BMT) by selectively expanding Tregs from 
co-administered syngeneic T cell-depleted marrow45,48.  Low-dose IL-2 also induces remission of 
autoimmune diabetes in NOD mice by selectively expanding Tregs and reducing pancreatic 
inflammation48,49; clinical trials of low-dose IL-2 for type 1 diabetes are currently recruiting or 
complete (ClinicalTrials.gov NCT01353833, NCT01827735).  Stallmach and colleagues showed 
that a fusion protein of IL-2 bound to IgG2b (IL-2IgG2b) prevents and ameliorates 2,4,6-
trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice49,50.  It was subsequently found that 
IL-2IgG2b caused Treg expansion.  We have generated preliminary data showing that TNBS 
colitis in “humanized” NOD-Scid-c-/- (NSG) mice, engrafted with human PB mononuclear cells 
(PBMCs), can be both prevented and ameliorated by low-dose human IL-2 (Goettel, Snapper, 
unpublished). 
 
Low-dose IL-2 has been shown to be both safe and effective in the treatment of GvHD.  
Some years ago, our collaborators safely administered low-dose IL-2 (0.2-0.6x106 IU/m2/day IV) 
to 29 patients who underwent CD6+-depleted allogeneic BMT following hematological 
malignancy, without GvHD, expanding both NK cells (for a graft vs. leukemia effect) and Tregs 
(preventing GvHD) in vivo, leading to improved disease-free survival50,51.  Recently, our 
collaborators showed that low-dose IL-2 (0.3-3x106 IU/m2/day SC) was safe and effective at 
treating steroid-resistant chronic GvHD following allogeneic hematopoietic stem cell 
transplantation (HSCT)43,51.  Low-dose IL-2 therapy was associated with a sustained increase in 
circulating Tregs and a rapid reduction in Tcon activation, which correlated with clinical 
response43,51.   


 
 
 
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Low-dose IL-2 is also safe and effective in the treatment of HCV-associated vasculitis 
and safe in HIV.  Saadoun and colleagues used low-dose IL-2 (1.5x106 IU/day SC) to manage 
treatment-resistant HCV-associated vasculitis, resulting in selective Treg expansion with a 
reduction in marginal zone B cells and cryoglobuliemia in vivo without increased HCV viremia52.  
Low-dose IL-2 has also been safely used with the intention of boosting CD4+ T cells in HIV, 
primarily resulting in increased circulating Tregs53. 
4.6 
Safety Profile of Low Dose IL-2:  Side-Effects Seen in Recent Clinical Trials. 
 
Soiffer et al.50 administered low-dose IL-2 by continuous IV infusion to 29 patients who 
underwent CD6-depleted BMT at least 6 weeks previously, with the intention of boosting Graft 
vs. Leukemia activity.  Doses ranged from 0.2 to 0.6x106 IU/m2/day.  25/29 patients completed 4 
weeks of therapy, while 17/29 patients completed at least 8 weeks of therapy.  Observed 
toxicities included fatigue (52%), temperature >38C (48%), nausea/vomiting (38%), myalgias 
(31%), diarrhea (24%), edema/weight gain (21%), dyspnea (21%), catheter infection (17%), 
thyroid dysfunction (7%), thrombocytopenia (<20,000; 7%) and neutropenia (<500; 3%).  
Toxicities were most prominent in patients on higher doses.  
 
In contrast to IV administration, SC administration is associated with slower absorption 
with maximum concentrations occurring 2-5h after dosing, 20-30-fold lower peak serum levels 
and a longer elimination half-life (5h SC vs. 1.5h IV)40, all of which may contribute to a favorable 
toxicity profile with SC administration. 
 
Koreth et al.51 administered daily SC low-dose IL-2 to 29 patients (28 were evaluable) 
with steroid-resistant chronic GvHD following HSCT.  The maximum tolerated dose (MTD) in this 
study was found to be 1x106 IU/m2/day.  The higher dose of 3x106 IU/m2/day was associated 
with persistent grade 1 constitutional symptoms, necessitating dose reduction.  Toxicities related 
to IL-2 included reversible induration of SC injection sites (n=3/28), grade 2 constitutional 
symptoms (fever, malaise and fatigue in 1/28), grade 2 renal dysfunction (1/28) and grade 2 
thrombocytopenia (1/28).  Two patients in this study developed thrombotic microangiopathy-
associated renal failure requiring dialysis.  Both of these patients received sirolimus and 
tacrolimus, which are associated with thrombotic microangiopathy.  Three patients developed 
grade 3 or grade 4 infections.  One patient with a background of MRSA pneumonia developed 
recurrent MRSA pneumonia following 7 weeks of therapy.  A second patient developed 


 
 
 
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Hemophilus influenzae type-B bacteremia following 4 weeks of treatment.  A third patient had a 
pre-existing MRSA furuncle, necessitating discontinuation of treatment at day 2. 
 
While the study design of this proposed phase Ib/IIa clinical trial is based on Koreth et 
al’s study, patients with active UC are likely to be fitter than patients with steroid-resistant GvHD 
following BMT.  In a recent study with a more comparable group, Saadoun et al.52 treated 10 
patients with HCV-associated vasculitis that was unresponsive to conventional anti-viral therapy 
and/or rituximab with low-dose IL-2: 1.5 MIU/day for 5 days, followed by three 5-day courses of 3 
MIU/day at weeks 3, 6 and 9.  Only mild (grade 1) toxicities were observed, including fatigue 
(4/10), flu-like symptoms (4/10), SC injection site reaction (2/10), arterial hypertension (1/10) and 
myalgia (1/10). 
4.7 
Hypothesis. 
 
Based on these data, we hypothesize that low-dose IL-2 will safely expand PB and 
LP Tregs in vivo in patients with UC and be efficacious in controlling disease. 
 
 


 
 
 
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5 
STUDY DESIGN AND OBJECTIVES. 
5.1 
Study Design. 
 
An open-label, phase Ib/IIa clinical trial, with a 3+3 dose escalation design, to determine 
the safety and the maximum tolerated dose (MTD) of daily SC recombinant human interleukin-2 
(IL-2, aldesleukin, Proleukin®) in adult subjects with moderate to severe UC. 
5.2 
Primary Objectives. 
(i). 
To determine the safety of daily SC low dose IL-2 in subjects with moderate to severe UC 
(ii). 
To determine the MTD of daily SC low dose IL-2 in subjects with moderate to severe UC. 
5.3 
Secondary Objectives. 
(i). 
To determine the feasibility of administering an eight week course of daily SC low dose IL-
2 to subjects with moderate-to-severe UC. 
(ii). 
To determine the clinical response to the above treatment. 
(iii). To assess the immunological response to the above treatment, with particular reference to 
numbers and function of Tregs in the blood and intestinal lamina propria (LP) 
 
 


 
 
 
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6 
SUBJECT SELECTION. 
6.1 
Inclusion Criteria. 
(i). 
Age 18-70 years.  Maximum age limit for subjects recruited at BCH will be 30 years.  
(ii). 
A diagnosis of UC made by standard clinical, radiological, endoscopic and histological 
criteria. 
(iii). Moderate to severe UC with a Mayo score of 6-12. 
(iv). Failure to tolerate or failure to respond to at least one conventional therapy with the 
intention of inducing or maintaining remission (examples include oral corticosteroids, oral 
5-aminosalicylates, azathioprine and/or 6-mercaptopurine, or a TNF antagonist).  
Corticosteroid dependency (inability to taper oral corticosteroids without a recurrence of 
disease activity) is also included in this category. 
(v). 
Stable doses of concomitant medications, as defined in Section 7 (page 19). 
(vi). A negative pregnancy test in the 2 weeks prior to anticipated commencement of the study 
drug, in female subjects of child-bearing age.  Men and women of reproductive potential 
must agree to use an acceptable method of birth control during treatment and for six 
months after completion of treatment. 
(vii). Ability to provide informed consent.  
 
 


 
 
 
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6.2 
Exclusion Criteria. 
(i). 
A diagnosis of Crohn’s disease or indeterminate colitis. 
(ii). 
Requirement for immediate surgical, endoscopic or radiological intervention for toxic 
megacolon, massive hemorrhage, perforation, sepsis, or intra-abdominal or perianal 
abscess.  
(iii). Ileostomy, proctocolectomy or subtotal colectomy with ileorectal anastomosis. 
(iv). History of colorectal cancer or dysplasia. 
(v). 
Positive stool test for Clostridium difficile. 
(vi). Current medically significant infection. 
(vii). Significant laboratory abnormalities;  
a. 
Hb < 8.0 g/dL, WBC < 2.5 x 103/mm3, Plt < 100 x 103/mm3. 
b. 
Creatinine ≥ 1.5x institutional ULN. 
c. 
Total bilirubin > 2.0 mg/dL, ALT > 2x institutional ULN.  Elevated unconjugated 
bilirubin related to Gilbert’s syndrome is allowed. 
d. 
Abnormal thyroid function tests. 
(viii). Positive serology for HIV, hepatitis B virus (HBV) or hepatitis C virus (HCV). 
(ix). Positive screening test for tuberculosis (TB). 
(x). 
First dose of an anti-TNF medication within 4 weeks of anticipated study commencement, 
or a subsequent dose within 2 weeks of commencement; or ciclosporin or tacrolimus 
within 2 weeks of anticipated study commencement. 
(xi). Received another IND within 5 half-lives of that agent before the planned commencement 
of SC IL-2. 
(xii). Malignancy within the last 5 years. 


 
 
 
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(xiii). Allergy to any component of the study drug. 
(xiv). Pregnant or lactating women. 
(xv). Inability to comply with the study protocol or inability to give informed consent. 
(xvi). Prior exposure to IL-2. 
(xvii). Uncontrolled cardiac angina or symptomatic congestive cardiac failure (NYHA Class III or 
IV). 
 
 


 
 
 
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7 
RULES FOR CONCOMITANT IBD MEDICATIONS. 
(i). 
Subjects receiving oral 5-aminosalycilates or oral corticosteroids (prednisone ≤40mg per 
day or its equivalent) must receive stable doses for at least 2 weeks prior to 
commencement of the study. 
(ii). 
Subjects receiving azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, 
sirolimus or mycophenolate mofetil must receive stable doses for at least 4 weeks prior to 
commencement of the study. 
(iii). Subjects receiving concurrent tacrolimus and sirolimus will be excluded, due to the 
increased risk of renal failure seen in previous studies when these medications were co-
prescribed.   
(iv). Rectally administered steroids or mesalamine will be discontinued 2 weeks before 
screening.  
(v). 
Subjects receiving IV steroids or biologic agents must undergo a 4-week wash-out period 
prior to enrolment.  
 
 


 
 
 
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8 
SUBJECT ENTRY. 
8.1 
Recruitment and Accrual. 
 
Subjects enrolled in the trial will be identified through the gastroenterology department at 
Boston Children’s Hospital. 
8.2 
Study Site Responsible Investigator. 
Boston Children’s Hospital 
Scott B Snapper MD PhD 
Boston Children’s Hospital 
Division of Medicine (GI/Nutrition), 
300 Longwood Avenue, Enders 676, 
Boston MA 02115. 
Tel: (617) 919-4973 
 
 


 
 
 
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9 
TREATMENT PROGRAM. 
9.1 
Description of Study Design. 
 
This is an open-label phase Ib/IIa clinical trial to determine the safety and maximum 
tolerated dose of daily SC IL-2 in eligible subjects with moderate to severe ulcerative colitis 
(UC).  Subject cohorts will receive escalating dose levels of IL-2, as shown in Table 1 (page 22).  
A minimum of three subjects will be treated in each cohort, with an additional ten subjects 
treated at the presumptive MTD. 
9.2 
Dose Cohorts. 
 
Koreth et al.51 treated subjects with steroid-resistant GvHD with doses ranging from 0.3-
3.0x106 IU/m2/day.  Doses of 0.3 and 1.0x106 IU/m2/day were well-tolerated.  1.0x106 IU/m2/day 
was found to be the MTD in this study.  Dose limiting toxicity (DLT) occurred at 3.0x106 
IU/m2/day.  Saadoun et al.52 treated subjects with treatment-resistant HCV-associated vasculitis 
at a dose of 1.5x106 IU/day, which was well-tolerated in this study.  The side-effects seen in 
these studies are detailed in Section 4.6 (page 13). 
 
Dose level A:  0.3x106 IU/m2/day.  Koreth et al.51 found that a dose of 0.3x106 IU/m2/day 
was well tolerated in a cohort of subjects with steroid-resistant GvHD.  Consequently, we 
propose to commence treatment at this dose.   
 
Dose level B:  1x106 IU/m2/day.  Provided dose level A is tolerated, the dose will then 
be escalated to dose level B.  This was the MTD in the Koreth et al. study51. 
 
Dose level C:  1.5x106 IU/m2/day.  Intolerable constitutional symptoms occurred at the 
highest dose in the Koreth et al. study51 (3x106 IU/m2/day).  Consequently, we propose that the 
highest dose in this study will be 1.5x106 IU/m2/day, or 50% of the dose at which intolerable 
constitutional symptoms occurred in that study.  This dose has been included to maximize the 
potential for clinical response, while minimizing the potential for toxicity.  This dose remains two 
orders of magnitude below those used in studies of IL-2 for metastatic melanoma and RCC40. 
 
 


 
 
 
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Table 1: Dose Escalation Cohorts. 
 
Cohort 
IL-2 Dose 
(IU/m2/day) 
Projected n in 
each cohort 
Dose Level A 
0.3 x 106 
3 
Dose Level B 
1 x 106 
3 
Dose Level C 
1.5 x 106 
3 + 3 + 10* 
*Assuming dose level C is shown to be the MTD. 
Adapted from Koreth et al.51 
9.3 
Dose Escalation Schema. 
There will be 3 dose levels, ranging from 0.3 to 1.5x106 IU/m2/day.  Each subject 
will be treated at a single dose level.  The first three subjects will be enrolled at the lowest 
dose level:  dose level A.  These subjects will be treated sequentially, so that none of 
these subjects will receive the study drug concurrently.  The dose will be escalated as per 
the protocol below.   
The maximum tolerated dose (MTD) is the highest tolerated dose level at which a 
minimum of 6 subjects have been evaluated, with fewer than 2 evaluable subjects in 6 
experiencing a dose limiting toxicity (DLT); i.e. DLT in 1/6 evaluable subjects.   
 
A dose escalation schema has been designed in order to identify the MTD. 
9.3.1 Dose Level A. 
Three evaluable subjects will be recruited at dose level A.  They will be monitored for the 
development of DLT during their 8-week treatment course.  


 
 
 
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If none (0/3) of these subjects experience a DLT during the 8-week treatment, dose 
escalation will occur so that the next three subjects will be enrolled at dose level B. 
If one of the first three subjects treated (1/3) at dose level A experiences a DLT during 
the 8-week treatment, an additional 3 subjects will be enrolled at dose level A.  Up to six 
subjects in total will then complete treatment at dose level A.  If no further DLTs occur in this 
group of 6 subjects (i.e. 1/6 subjects with DLT), dose escalation will occur so that the next three 
subjects will be enrolled at dose level B.  If DLTs occur in two subjects in this group (i.e. 2/6 
subjects with DLT), then dose level A has not been tolerated and the study will be terminated 
early.  
If two of the first three subjects treated (2/3) at dose level A experience DLT during the 8-
week treatment, then dose level A has not been tolerated and the study will be terminated early. 
The dose may be reduced from dose level B to dose level A.  If this occurs and six 
subjects have already been treated at dose level A, then dose level A is the MTD.  If this occurs 
and only three subjects have been treated at dose level A, then a further three subjects will be 
recruited at dose level A.  If 0 or 1 subject in this group of 6 experience a DLT, then dose level A 
will be the MTD.  If 2 subjects in this group of 6 experience a DLT, then the study will be 
terminated early. 
9.3.2 Dose Level B. 
 After successful dose escalation from dose level A, the next three evaluable subjects will 
be treated at dose level B.  They will be monitored for the development of DLT during their 8-
week treatment course.  
If none (0/3) of these subjects experience a DLT during the 8-week treatment, dose 
escalation will occur so that the next three subjects will be enrolled at dose level C. 
If one of the first three subjects treated at dose level B (1/3) experiences a DLT during 
the 8-week treatment, an additional 3 subjects will be enrolled at dose level B.  Up to six 
subjects in total will then complete treatment at dose level B.  If no further DLTs occur in this 
group of 6 subjects (i.e. 1/6 subjects with DLT), dose escalation will occur so that the next three 
subjects will be enrolled at dose level C.  If DLTs occur in two subjects in this group (i.e. 2/6 
subjects with DLT), then dose level B has not been tolerated and the dose will be reduced to 
dose level A (see Section 9.3.1, page 22).  


 
 
 
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If two (2/3) of these subjects experience a DLT during the 8-week treatment, then dose 
level B has not been tolerated and the dose will be reduced to dose level A  (see Section 9.3.1, 
page 22). 
The dose may be reduced from dose level C to dose level B.  If this occurs and six 
subjects have already been treated at dose level B, then dose level B is the MTD.  If this occurs 
and only three subjects have been treated at dose level B, then a further three subjects will be 
recruited at dose level B.  If 0 or 1 subject in this group of 6 experience a DLT, then dose level B 
will be the MTD.  If 2 subjects in this group of 6 experience a DLT, then the dose will be reduced 
from dose level B to dose level A. 
 
9.3.3 Dose Level C. 
After successful dose escalation from dose level B, the next three evaluable subjects will 
be treated at dose level C.  They will be monitored for the development of DLT during their 8-
week treatment course.  
If none or one (0-1/3) of the first three subjects recruited at dose level C experience a 
DLT during the 8-week treatment, a further three subjects will be recruited at this dose level.  Up 
to six subjects in total will then complete treatment at dose level C.  If no further DLTs occur in 
this group of 6 subjects (i.e. 0-1/6 subjects with DLT), dose level C will be the MTD.  If DLTs 
occur in two subjects in this group (i.e. 2/6 subjects with DLT), then dose level C has not been 
tolerated and the dose will be reduced to dose level B (see Section 9.3.2, page 23).  
If two of the first three subjects treated at dose level C (2/3) experience a DLT during the 
8-week treatment, then dose level C has not been tolerated and the dose will be reduced to 
dose level B (see Section 9.3.2, page 23).  
 
The dose escalation schema is summarized in Table 2 (page 25). 
9.3.4 Recruitment of Additional Subjects. 
 
An additional 10 subjects will be treated at the MTD to further characterize the safety and 
efficacy of the study drug per the definition of MTD above. 
 


 
 
 
Page 25 of 56 
 
Table 2: Rules for Dose Escalation and Allowable DLTs Per Cohort 
# DLT per cohort 
Dose escalation rule 
0/3 
• Accrue the next 3 subjects at next dose level 
• If dose level C, accrue 3 additional subjects at this level 
1/3 
• Accrue 3 additional subjects at current dose level 
2/3 
• Current dose level has not been tolerated.  Reduce dose to the lower 
dose level 
o 
If only 3 subjects have been treated at the lower dose level, 
accrue an additional 3 subjects at the lower dose level. 
o 
If 6 subjects have already been treated at the lower dose level, 
the lower dose level is the MTD. 
o 
If this occurs at dose level A, the study will be terminated early. 
1/3 + 0/3 (1/6) 
• Escalate dose. Accrue 3 new subjects at next highest dose level.  If 
0/6 or 1/6 DLTs at dose level C, then this is the MTD. 
1/3 + 1/3 (2/6) 
• Current dose level has not been tolerated.  Reduce the dose.  If this 
occurs at the lowest dose level, the study will be terminated early. 
To be read with reference to Section 9.3 (page 22), above.  Adapted from Koreth et al.51 
 
9.4 
Determination of Maximum Tolerated Dose. 
 
The maximum tolerated dose (MTD) is the highest tolerated dose level at which a 
minimum of 6 subjects have been evaluated, with fewer than 2 evaluable subjects in 6 
experiencing a dose limiting toxicity (DLT); i.e. DLT in 1/6 evaluable subjects. In addition to 
the above, at least 1 patient should meet the criteria for response or remission for it to be 
considered the MTD.  


 
 
 
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Subjects will be considered unevaluable for DLT if they are removed from the study or 
die without developing DLT prior to receiving at least 8 weeks of IL-2 therapy.  If there are fewer 
than 3 evaluable subjects in a cohort, additional subjects will be added at that dose level. 
 
Intolerable lower grade toxicity that does not meet the definition of DLT, but still means 
that a subject is unable to complete the per-protocol treatment will be taken as equivalent to a 
DLT for the purpose of assessing dose escalation.  If 2 subjects in a cohort of 3 refuse to 
continue at that dose level for at least 8 weeks due to intolerable lower grade toxicity, that dose 
level will not be regarded as the MTD.  
9.5 
Definition of Dose Limiting Toxicities. 
 
Organ toxicities will be assessed at scheduled clinical visits and graded according to the 
National Cancer Institute Common Terminology Criteria for Adverse Events v4.054. 
(i). 
Anaphylaxis:  Life threatening anaphylaxis due to the study drug is considered a DLT.  
(ii). 
Grade 4 toxicity:  Grade 4 toxicity (except colitis, see below) directly related to IL-2 by 
week  8 of IL-2 treatment is considered a DLT.  
(iii). 
Grade 3 toxicity:  IL-2 will be withheld for grade 3 toxicity (except colitis). 
(iv). 
Development of a life-threatening infection:  Development of a life threatening 
infection, as assessed by the treating physician, prior to week 8 of IL-2 therapy will be 
considered a DLT.  IL-2 will be withheld. 
(v). 
Grade 2 renal toxicity: IL-2 will be withheld in the event of a grade 2 renal toxicity 
(creatinine = 1.5-3.0 x ULN). 
Development of toxic megacolon or colitis requiring immediate surgical intervention is a well-
described risk of active UC.  Consequently, it will not be considered a DLT.  However, IL-2 will 
be discontinued. 


 
 
 
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9.6 
Clinical Assessments for Toxicity. 
 
Subjects will undergo regular clinical examination (history and physical examination), 
blood tests and other tests as indicated at scheduled visits in order to detect potential drug 
toxicity, as detailed in Section 11 (Page 31). 
9.7 
Extended Treatment with Low-Dose IL-2. 
 
After completing 8 weeks of IL-2 therapy, subjects who have achieved a clinical response 
or entered remission (see Section 14, Page 43, for definitions) at any dose level and have an 
acceptable toxicity profile will be allowed to continue treatment at that dose level, at the 
discretion of the treating physician.  These subjects will be seen in clinic every 3 months for a 
year, after which they will be followed by the referring gastroenterologist.  Prometheus 
Laboratories will continue to supply IL-2 for up to one year after completing 8 weeks of IL-2 
therapy to subjects who have achieved a clinical response or entered remission at any dose 
level and have an acceptable toxicity profile. 
 
Subjects on extended duration IL-2 therapy will not be evaluable for toxicity and DLT 
endpoints.  The addition of other medications (except the combination of sirolimus and 
tacrolimus) will be permitted at the discretion of the treating physician.  Subject assessment 
while on extended treatment with IL-2 is detailed in Section 11.4 (page 34).  In the event of 
toxicity attributable to IL-2, therapy will be discontinued.   
 
 


 
 
 
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10 
STUDY DRUG: ALDESLEUKIN. 
10.1 
Formulation, Reconstitution and Storage. 
 
Recombinant human IL-2 (aldesleukin, Proleukin®) is supplied free of charge by 
Prometheus Laboratories, Inc. (San Diego, CA). 
 
Aldesleukin is supplied as a sterile, white to off-white, lyophilized cake in single-use vials 
containing 22 million international units (MIU) of aldesleukin intended for intravenous (IV) 
administration.  Vials of lyophilized aldesleukin will be stored in a refrigerator at 2-8°C (36-46°F) 
and will not be used beyond the expiration date printed on the label. 
 
On reconstitution of a 22 MIU vial of aldesleukin with 1.2 ml of sterile water for injection 
(SWFI), each ml contains 18 MIU (1.1mg) aldesleukin, 50 mg mannitol and ~180 mcg sodium 
dodecyl sulphate, buffered with ~170 mcg sodium phosphate monobasic and 890 mcg sodium 
phosphate dibasic to a pH of 7.5 (range: 7.2-7.8). 
10.2 
Reconstitution. 
 
Each 22 MIU vial of aldesleukin should be reconstituted with 1.2 ml of sterile water for 
injection (SWFI).  During reconstitution the SWFI should be directed at the sides of the vial to 
avoid foaming, and the contents of the vial should be gently swirled.  THE VIAL SHOULD NOT 
BE SHAKEN. 
 
Reconstitution or dilution with Bacteriostatic Water for Injection or 0.9% Sodium Chloride 
for Injection should be avoided due to increased aggregates. 
 
 After reconstitution, the resulting solution should be a clear, colorless to slightly yellow 
liquid.  Reconstitution and dilution procedures other than those described may alter the delivery 
and/or pharmacology of aldesleukin and are not recommended. 
 
All syringes should be prepared in the pharmacy of the relevant institution at the same 
time, after diluting a 22 MIU aldesleukin vial with 1.2ml SWFI and 4.8ml D5W (final IL-2 
concentration=3.6 MIU/mL), and any remaining product should be immediately discarded. 


 
 
 
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10.3 
Storage. 
 
Aldesleukin is an unpreserved sterile product.  The reconstituted and diluted solutions 
should be stored in a refrigerator at 2-8°C (36-46°F).  DO NOT FREEZE. 
 
When reconstituted and diluted according to directions, aldesleukin is stable for up to 48 
hours in plastic bags (e.g. PVC bags) when stored at refrigerated AND room temperatures (2-
25°C [36-77°F]). 
 
From a microbial point of view, the product should be used immediately.  Vials should be 
entered only once for reconstitution to minimize the chances of contamination.  If not used 
immediately, in-use storage times should normally not be longer than 24 hours at 2-8°C (36-
46°F), unless reconstitution has been performed under controlled and validated aseptic 
conditions in a laminar airflow hood. 
 
 Data support the stability and sterility of reconstituted diluted aldesleukin preparations 
(reconstituted with SWFI and further diluted with D5W); and the stability and sterility of product 
reconstituted with SWFI but not further diluted, for up to 14 days at 2-8°C (36-46°F) when 
syringes are prepared by qualified health-care professionals under aseptic conditions40.  
Therefore, if reconstitution and dilution are performed under controlled and validated conditions 
using a laminar flow hood, the dose or doses thus prepared and stored at 2-8°C (36-46°F) need 
to be used within 14 days. 
 
On 4/26/2016, the Investigational Drug Service (IDS) at Brigham & Women’s Hospital, 
Boston MA, reduced the beyond use dating for aldesleukin that is prepared at their research 
pharmacy for this study from 14 days to 9 days.  Consequently, aldesleukin prepared at BWH 
IDS and subsequently stored at 2-8°C (36-46°F) needs to be used within 9 days. 
10.4 
Dosage. 
 
Aldesleukin will be given according to the dose schema described in Section 9.2 (page 
21), based on the cohort into which they are accrued.  The first two subjects to receive IL-2 will 
be admitted overnight to the Clinical Translational Study Unit (CTSU).  Aldesleukin may be 
administered in the home or out-subject setting by daily subcutaneous injection for 8 weeks.  
Subjects who receive additional treatment after experiencing clinical benefit from 8 weeks of 


 
 
 
Page 30 of 56 
initial therapy may continue aldesleukin therapy at their initial dose level.  They will be followed 
by their physician according to Section 9.7 (page 27). 
10.5 
Administration. 
 
The product should be inspected visually for particulate matter or discoloration and 
brought to room temperature before administration.  Administer as a subcutaneous injection.  
DO NOT ADMINISTER AS INTRAVENOUS PUSH OR BOLUS.  Premedications are not 
required prior to the first dose or escalated doses. 
 
 


 
 
 
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11 
CLINICAL AND LABORATORY EVALUATIONS. 
 
Participants will have 10 face-to-face encounters with research staff throughout the 
clinical trial, including (i) Screening at week -2, (ii) Baseline assessment prior to first dose (day 
0), (iii) Clinical assessment prior to second dose (day 1), (iv-viii) Assessments at day 4 optional 
visit) and weeks 1, 2, 4 and 6, (ix) End of treatment assessment at week 8, and (x) End of study 
assessment at week 12.  On completion of the study, research staff will contact participants by 
telephone monthly for 2 months and on a 2-monthly basis for a further two months.  
Assessments after week 1 (as indicated in Table 3, page 37) will be scheduled for the planned 
day 3 days.  Tests that would be performed as part of routine clinical care are highlighted as 
follows: “(routine)”. 
11.1 
Screening (Pre-Treatment) Evaluation (Week -2).  
 
Following signed, informed consent, the following screening evaluation will be performed 
within two weeks of the anticipated start date of aldesleukin therapy: 
• 
Medical history including confirmation of fulfillment of inclusion criteria (routine). 
• 
Physical examination, including vital signs, weight, height (routine). 
• 
Pregnancy test for women of childbearing potential. 
• 
Screening test for tuberculosis (TB):  IFN- Release Assay (IGRA), e.g. QuantiFERON®.  
If the IGRA result is “indeterminate” or “positive”, a CXR will be performed as part of 
routine clinical care to exclude pulmonary TB. 
• 
Stool for culture and sensitivity (C&S), ova and parasites (O&P), Clostridium difficile stool 
toxin and fecal calprotectin (routine). 
• 
Flexible sigmoidoscopy and biopsies with calculation of the Mayo score; or colonoscopy 
and biopsies if the subject has never had a full colonoscopy (routine).  As part of routine 
clinical evaluation, two biopsies will be obtained when possible from each of the following 
sites: rectum, sigmoid colon, descending colon (n=up to 6). In addition, biopsies for 
research will be obtained from each site. From the descending colon, biopsies will be 
obtained to characterize lamina propria immune cells on fresh cells by flow cytometry 


 
 
 
Page 32 of 56 
(n=2), transcriptomics (n=1), and for deeper immunologic and single cell analysis (n=5). 
From the sigmoid colon, biopsies will be obtained to characterize LPMCs on fresh cells 
by flow cytometry (n=2), transcriptomics (n=1), to cryomold for histologic assessment 
(n=2), and for deeper immunologic and single cell analysis (n=5). From the rectum, 
biopsies will be obtained to characterize LPMCs on fresh cells by flow cytometry (n=2), 
transcriptomics (n=1), mucosal microbiota (n=2), to cryomold for histologic assessment 
(n=2), and for deeper immunologic and single cell analysis (n=5).  
• 
Hematology:  Complete blood count (CBC) and differential (routine). 
• 
Inflammatory markers:  ESR and CRP (routine). 
• 
Serum chemistries:  glucose, BUN, creatinine, uric acid, total bilirubin, 
alkaline phosphatase, LDH, total protein, albumin, AST, ALT, and calcium (routine). 
• 
Thyroid function tests (TSH, T3, free T4) (routine). 
• 
Virology:  serology for HIV, HBV and HCV. 
Note: The following basic science investigations can be performed at the screening visit or at 
the baseline (Day 0) visit, at the discretion of the investigator.   
• 
Immunology bloods:  Quantitative flow cytometric assessment of leucocytes, NK cells 
and Treg subsets from PB and endoscopic biopsies.  Assessment of PB and LP Treg 
function.  Banking of PBMCs, plasma, RNA, DNA.   
• 
Microbiology:  Stool sample and mucosal biopsies for analysis of the intestinal 
microbiome. 
• 
IL-2 serum levels and anti-aldesleukin antibodies. 
11.2 
Baseline, Treatment and End-of-Study Evaluations (Day 0 to Week 6 and Week 12). 
 
The following evaluations will be performed at baseline (day 0), day 4 (optional visit), and 
weeks 1, 2, 4, 6 and 12: 


 
 
 
Page 33 of 56 
• 
Medical history and physical examination, including assessment of symptoms and signs 
in order to detect potential drug toxicity (routine).  
• 
Hematology:  Complete blood count (CBC) and differential (routine). 
• 
Inflammatory markers:  ESR and CRP (routine). 
• 
Serum chemistries:  glucose, BUN, creatinine, uric acid, total bilirubin, 
alkaline phosphatase, LDH, total protein, albumin, AST, ALT, and calcium (routine). 
• 
Thyroid function tests (TSH, T3, free T4) (week 12). 
• 
Immunology bloods:  Quantitative flow cytometric assessment of leucocytes, NK cells 
and Treg subsets from PB.  Assessment of PB and LP Treg function.  Banking of 
PBMCs, plasma, RNA, DNA. 
• 
Microbiology:  Stool sample for analysis of the intestinal microbiome (day 0, weeks 1, 2, 
4, 6, 8 and 12). 
• 
Fecal calprotectin (day 0, weeks 1, 2, 4, 6, 8 and 12). 
• 
IL-2 serum levels and anti-aldesleukin antibodies (not days 1 or 4). 
 
Participants will attend the CTSU for their first IL-2 dose on day 0 and their second IL-2 
injection on day 1.  Participants will be evaluated clinically at that encounter. 
11.3 
End of Treatment Evaluation (Week 8). 
 
The following evaluations will be performed at week 8: 
• 
Medical history and physical examination, including assessment of symptoms and signs 
in order to detect potential drug toxicity (routine).  
• 
Flexible sigmoidoscopy and biopsies with calculation of the Mayo score.  The same 
biopsies will be obtained as at the screening procedure, except that histology will be 
limited to the rectum and sigmoid/distal descending colon.   
• 
Hematology:  Complete blood count (CBC) and differential (routine). 


 
 
 
Page 34 of 56 
• 
Inflammatory markers:  ESR and CRP (routine). 
• 
Serum chemistries:  glucose, BUN, creatinine, uric acid, total bilirubin, 
alkaline phosphatase, LDH, total protein, albumin, AST, ALT, and calcium (routine). 
• 
Thyroid function tests (TSH, T3, free T4). 
• 
Immunology bloods:  Quantitative flow cytometric assessment of leucocytes, NK cells 
and Treg subsets from PB. Assessment of PB and LP Treg function. Banking of PBMCs, 
plasma, RNA, DNA. 
• 
Microbiology:  Stool sample and mucosal biopsies for analysis of the intestinal 
microbiome. 
• 
IL-2 serum levels and anti-aldesleukin antibodies. 
11.4 
Evaluation of Subjects Undergoing Extended Treatment with Low Dose IL-2. 
 
Patients with a treatment response who elect to undergo extended treatment (as 
described in Section 9.7, page 27) will be seen in clinic every 3 months as part of their routine 
clinical care.  Evaluation may include: 
• 
Medical history and physical examination, including assessment of symptoms and signs 
in order to detect potential drug toxicity (routine).  
• 
Hematology:  Complete blood count (CBC) and differential (routine). 
• 
Inflammatory markers:  ESR and CRP (routine). 
• 
Serum chemistries:  glucose, BUN, creatinine, uric acid, total bilirubin, 
alkaline phosphatase, LDH, total protein, albumin, AST, ALT, and calcium (routine). 
• 
Immunology bloods: Quantitative flow cytometric assessment of leucocytes, NK cells and 
Treg subsets from PB. Assessment of PB and LP Treg function. Banking of PBMCs, 
plasma, RNA, DNA. 


 
 
 
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11.5 
Details of Specific Assays.  
 
Routine laboratory tests will be performed at Boston Children’s Hospital, Labcorp or other 
facilities according to availability.   
 
The techniques required for immune phenotyping (e.g. FACS, PCR etc.) will be 
performed on appropriately collected samples at the Snapper Laboratory at Boston Children’s 
Hospital, the Harvard Digestive Disease Center and collaborating laboratories.  The laboratory 
personnel who will perform these assays will be identified and/or appointed prior to the inception 
of this study.  The specific protocols for each investigation, including the number of cells needed 
for each assay and precise locations and numbers of endoscopic biopsies etc., will be placed in 
the Trial Master File.  A maximum of 26 biopsies will be obtained from each participant at each 
endoscopy for the purpose of these investigations, as described in Section 11.1 (page 31).  
• 
Quantification of leucocytes and Treg subsets will be performed by flow cytometric 
identification of CD3, CD4, CD8, CD25, CD127, CD45RA and FOXP3 in PBMC and 
LPMC preparations.  CD16 and CD56 expression will be used to identify NK cells. 
• 
The biological effect of SC IL-2 on STAT5 phosphorylation in effector T cells and Tregs 
will be assessed by flow cytometry.  
• 
The suppressive function of PB and LP Tregs prior to and during treatment will be 
evaluated by assessment of Treg-mediated inhibition of CFSE dilution, using sorted 
CD4+CD25hiCD127lo Tregs and freeze-thawed CFSE-labelled allogeneic CD4+CD25- T 
responders. 
• 
Intestinal microbiome analysis will be performed on snap-frozen stool and endoscopic 
biopsy samples.  This will include phylogenetic identification and subtyping based on 16S 
ribosomal RNA classification and will be performed at the Harvard Digestive Disease 
Center Microbiome Core Facility.   
11.6 
Assessment of Aldesleukin Immunogenicity. 
 
 
IL-2 levels and anti-aldesleukin antibodies will be determined at baseline, weeks 1, 2, 4, 
6, 8 and 12.  If anti-aldesleukin antibodies are detected, anti-aldesleukin antibody levels will be 
monitored beyond the 12-week study period until antibody levels have returned to baseline.   


 
 
 
Page 36 of 56 
 
 


 
 
 
Page 37 of 56 
Table 3: Summary of Required Data 
 
Screening 
Days 0, 1a, 4b 
Week 
Week  
(Pre-treatment) 
Weeks 1, 2, 4, 6 
 8 
12 
Medical history 
  
  
  
  
Physical examination 
  
  
  
  
Pregnancy test 
  
 
 
 
Viral testing: HIV, HBV, HCV 
  
 
 
 
Screening for TB 
  
 
 
 
Stool for C&S, O&P, C. difficile 
  
 
 
 
Colonoscopy/flexible sigmoidoscopy1 & 
Mayo score 
  
 
  
 
CBC & differential 
  
 * 
  
  
ESR, CRP 
  
 * 
  
  
Serum chemistry 
  
 * 
  
  
Thyroid function tests 
  
 
  
  
IL-2 serum levels 
  
 * 
  
  
Anti-aldesleukin antibodies 
  
 2 
  
  
Intestinal microbiome analysis4 
  
 2 
  
  
Fecal calprotectin 
  
 3 
  
  
Immune phenotyping 
  
 * 
  
  
Notes. a: History and physical examination only at day 1, unless additional investigations indicated 
clinically.  b: Day 4 visit is optional. 
1: A full colonoscopy may be performed at screening to define disease extent in addition to mucosal 
inflammation if the subject has never had a full colonoscopy.  
2: Not days 1 or 4.  3. Not day 1. 
 
 


 
 
 
Page 38 of 56 
11.7 
Anticipated Toxicities. 
 
The toxicities seen in clinical trials utilizing similar doses of IL-2 are shown in Table 4, 
below.  They are also discussed in Section 4.6 (page 13). 
Table 4:  Anticipated Toxicities 
Study 
IL-2 dose 
Indication 
Evaluable 
subjects (n) 
Observed toxicities 
n (%) 
Soiffer 
et al.50 
0.2-0.6x106 
IU/m2/day 
IV 
Enhance 
graft vs. 
leukemia 
effect 
following 
HSCT 
29 
Fatigue 
15 (52) 
Temp >38°C 
14 (48) 
Nausea/vomiting 
11 (38) 
Myalgia 
9 (31) 
Rash 
9 (31) 
Diarrhea 
7 (24) 
Edema 
6 (21) 
Dyspnea 
6 (21) 
Catheter infection 
5 (17) 
Thyroid dysfunction 
2 (7) 
Thrombocytopenia 
2 (7) 
Koreth 
el al.51 
0.3-3x106 
IU/m2/day 
SC 
Treatment of 
steroid-
resistant 
GvHD 
28 
Injection site induration 
3 (11) 
Infections* 
3 (11) 
Renal failure** 
2 (7) 
Renal dysfunction 
1 (4) 
Flu-like symptoms 
1 (4) 
Thrombocytopenia 
1 (4) 
Saadoun 
et al.52 
1.5-3x106 
IU/day 
SC 
Treatment of 
HCV-
associated 
vasculitis 
10 
Fatigue 
4 (40) 
Flu-like symptoms 
4 (40) 
Injection site induration 
2 (20) 
Arterial hypertension 
1 (10) 
Myalgia 
1 (10) 
*  Recurrent MRSA pneumonia (1), Hemophilus influenzae type-B bacteremia (1), MRSA furuncle (1) 
** Thrombotic microangiopathic renal failure in two subjects taking tacrolimus and sirolimus.  This is 
a recognized complication of this combination of medications. 
 
 


 
 
 
Page 39 of 56 
11.8 
Criteria for Treatment Discontinuation. 
 
Subjects will be removed from the study and therapy with IL-2 should be abandoned for 
any of the following circumstances if they are deemed possibly, probably or definitely related to 
IL-2, or per subject preference: 
• 
The subject withdraws his/her consent at any time.  
• 
Anaphylactic reaction to IL-2.  
• 
Life threatening infection on IL-2.  
• 
Other grade 4 toxic event. 
• 
Grade 3 toxicity.  
• 
Grade 2 renal toxicity. 
• 
The development of toxic megacolon or colitis requiring immediate surgical intervention. 
• 
At the discretion of the treating physician. 
11.9 
Dose Modification/Toxicity Management. 
In the event of IL-2 related toxicity that does not meet the requirement for treatment 
discontinuation (grade 2 toxicity or less, excluding grade 2 renal toxicity), the treating physician 
may elect to withhold IL-2 to allow the toxicity to resolve.  IL-2 can be recommenced at 50% of 
the target dose if the toxicity resolves to grade 1 or better within 2 weeks.  At the discretion of 
the treating physician, if re-introduction of IL-2 is tolerated by the subject, the dose of can be 
escalated to the target dose.  
 
 


 
 
 
Page 40 of 56 
12 
REPORTING OF UNANTICIPATED PROBLEMS INVOLVING RISKS TO RESEARCH 
SUBJECTS, INCLUDING ADVERSE EVENTS. 
12.1 
Reporting to the Principal Investigator. 
 
The following events will be reported to the principal investigator within 24 hours of being 
known:  
• 
Death of a research subject. 
• 
Dose-limiting toxicity. 
• 
Adverse event that is unexpected and related or possibly related to the subject’s 
participation in research. 
• 
Medication or laboratory errors.  
• 
Breach of confidentiality or HIPAA violation. 
• 
Significant protocol deviation or non-compliance. 
• 
A research-related complaint. 
• 
Intentional change to protocol without IRB approval, to eliminate apparent immediate 
hazard to research subject.  
• 
Enforcement action. 
• 
Study personnel misconduct. 
• 
Incarceration of a research subject during study participation. 
• 
Any other event that the investigator thinks may represent an unanticipated problem that 
involves risks to subjects or others.  
 
 


 
 
 
Page 41 of 56 
12.2 
Reporting to the Institutional Review Board. 
 
Consistent with institutional guidance, the principal investigator will use a CHeRP 
“Unanticipated Problem or Event Form” to report the following events to Boston Children’s 
Hospital’s Committee on Clinical Investigation within 72 hours of being known:  
• 
Death of a research subject, thought to be related to the research study or possibly 
related to the research study. 
• 
Adverse event that is both unexpected and related or possibly related to the subject’s 
participation in research. 
• 
Medication or laboratory errors.  
• 
Breach of confidentiality or HIPAA violation. 
• 
Significant protocol deviation or non-compliance. 
• 
A research-related complaint. 
• 
Intentional change to protocol without IRB approval, to eliminate apparent immediate 
hazard to research subject.  
• 
Interim findings, publication or safety report. 
• 
Enforcement action. 
• 
Study personnel misconduct. 
• 
Incarceration of a research subject during study participation, so that additional IRB 
review can be accomplished in order for the participant to remain in the study. 
• 
Any other event that the investigator thinks may represent an unanticipated problem that 
involves risks to subjects or others. 
 
 


 
 
 
Page 42 of 56 
12.3 
Reporting to Prometheus Laboratories. 
 
Prometheus Laboratories will supply the study drug free-of-charge.  The principal 
investigator will transmit an anonymized copy of any serious adverse event reports (or FDA 
Form 3500A MedWatch reports, if applicable) to Prometheus Laboratories.  
 
Prometheus Drug Safety Email: 
drugsafety@prometheuslabs.com 
 
Prometheus Drug Safety Fax: 
(858) 754-3046 
 
13 
DATA SAFETY AND MONITORING BOARD. 
 
A Data Safety and Monitoring Board (DSMB) has been constituted.  The DSMB 
members are as follows:  Hans Herfarth MD PhD (Chair, Gastroenterologist, University of North 
Carolina at Chapel Hill),  Ashwin Ananthakrishnan MB BS MPH (Gastroenterologist, 
Massachusetts General Hospital), and Tanya Logvinenko PhD (Statistician, Boston Children’s 
Hospital).  The DSMB will determine stopping points for individual subjects and the study as a 
whole.  In addition, the DSMB will determine the schedule of meetings in order to review subject 
safety information and will also make recommendations concerning the continuation, 
modification or termination of the study.   
 
The DSMB will review study data and make recommendations on the conduct of the 
study at the following time points:  
• 
In the event of any serious adverse events or DLT. 
• 
When the third subject completes 8 weeks of IL-2 treatment, as per protocol. 
• 
Three months after study initiation. 
• 
Six months after study initiation. 
• 
At six month intervals thereafter. 
 
 


 
 
 
Page 43 of 56 
14 
STUDY ENDPOINTS. 
 
Subjects will be periodically assessed as described in Section 11 (page 31) in order to 
determine if the primary and secondary endpoints have been met. 
14.1 
Determination of Safety of Daily SC Low Dose IL-2. 
 
The safety of daily low-dose IL-2 will be determined by assessing for potential side-
effects and dose limiting toxicity at each study visit.   
14.2 
 Determination of the Maximum Tolerated Dose of Daily SC Low Dose IL-2. 
 
The MTD will be determined using the dose escalation strategy and assessments 
described in Sections 9 and 11 (pages 21 and 31, respectively). 
14.3 
Determination of the Feasibility of Administering an Eight Week Course of Daily SC 
Low Dose IL-2 in Subjects with Moderate to Severe UC. 
 
This relates to the technical feasibility of administering daily low-dose SC IL-2 as per 
protocol.  The narrative report of this outcome will include the numbers of subjects enrolled, 
subjects who completed the protocol and a description of any barriers to the technical feasibility 
of this approach in subjects with UC. 
14.4 
Determination of the Clinical Response to Daily SC Low Dose IL-2. 
 
Mayo score7,55 (Table 5; page 44) will be assessed as per protocol between weeks -2 
and 0 (as close to week 0 as possible) and week 8 in order to determine clinical response.  
Moderate to severe UC, denoted by a Mayo score of 6-12 is an inclusion criterion.  Clinical 
response and remission are defined according to Rutgeerts et al.7 
14.4.1 Definition of Clinical Response. 
 “A decrease from baseline in the total Mayo score of at least 3 points and at least 30% , 
with an accompanying decrease in the subscore for rectal bleeding of at least 1 point or 
an absolute subscore for rectal bleeding of 0 or 1.” 7 


 
 
 
Page 44 of 56 
14.4.2 Definition of Remission.. 
“A total Mayo score of 2 points or lower, with no individual subscore exceeding 1 point.”7 
14.4.3 Definition of Mucosal Healing. 
“An absolute subscore for endoscopy of 0 or 1.”7 
14.5 
Assessment of the Immunological Response to IL-2 Therapy. 
 
Data obtained from research laboratory-based assessment of subject immune responses 
to IL-2 therapy, that do not have direct clinical relevance, will be reported as appropriate for each 
data type. 
Table 5:  Mayo Scoring System for Assessment of Ulcerative 
Colitis Activity. 7,55 
Stool frequency* 
 
0 
Normal no. of stools for this subject 
 
1 
1 to 2 stools more than normal 
 
2 
3 to 4 stools more than normal 
 
3 
5 or more stools more than normal 
Rectal bleeding** 
 
0 
No blood seen 
 
1 
Streaks of blood with stool less than half the time  
 
2 
Obvious blood with stool most of the time 
 
3 
Blood alone passes 
Findings on endoscopy 
 
0 
Normal or inactive disease 
 
1 
Mild disease (erythema, decreased vascular pattern, 
mild friability)  
 
2 
Moderate disease (marked erythema, lack of vascular 
pattern, friability, erosions)  
 
3 
Severe disease (spontaneous bleeding, ulceration) 
Physician’s global assessment 
 
0 
Normal 
 
1 
Mild disease 
 
2 
Moderate disease  


 
 
 
Page 45 of 56 
 
3 
Severe disease 
 
 
*  Each subject serves as their own control. 
**  Represents the most severe bleeding of the day. 
 
 


 
 
 
Page 46 of 56 
15 
STATISTICAL CONSIDERATIONS. 
 
The primary endpoint of this Phase I study is to determine the MTD of daily 
subcutaneous IL-2 in moderate to severe UC. 
15.1 
Probability of Dose Escalation Under Various DLT Rates. 
 
Table 6 shows the probability of escalation under various true DLT rates.  With this 
design, there is 91% probability of escalation if the true rate of DLT is 10% and 17% probability 
of escalation if the true DLT rate is 50%. 
 
Table 6: Probability of Dose Escalation 
True rate of DLT 
Probability of dose escalation 
10% 
0.91 
20% 
0.71 
30% 
0.49 
40% 
0.31 
50% 
0.17 
60% 
0.08 
 
 
Once the MTD is established, an additional 10 evaluable subjects will be treated at the 
MTD level to further evaluate the toxicity as well as the feasibility.  The estimation of toxicity rate 
will be based on these 10 additional subjects. With 10 subjects, the 90% confidence interval of 
the toxicity rate will be within ±26%. 
15.2 
Accrual. 
 
We anticipate that the sample size will range from 3-28 evaluable subjects, depending on 
number of dose levels will be tested.  Based on the current practice, we anticipate that the 
accrual rate will be approximately 12 subjects per year, thus requiring up to 28 months to 
complete accrual. 
 


 
 
 
Page 47 of 56 
16 
ETHICS AND GOOD CLINICAL PRACTICE. 
 
This study will be performed in accordance with Boston Children’s Hospital’s policies and 
guidance on the conduct of clinical research, the principles of Good Clinical Practice (GCP), the 
Declaration of Helsinki and applicable state and federal laws and regulations.  
 
The study may not start without written Institutional Review Board approval and the 
written informed consent of the subject. 
 


 
 
 
Page 48 of 56 
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1987;317(26):1625–9.  
 


 
 
 
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18 
APPENDIX A:  MAYO SCORING WORKSHEET. 
 
Circle only one of each subscore and enter in equation below.  The worst diary entry from 
the three days prior to each study visit will be used for each subject-reported subscore. 
 
1. Stool Frequency Subscore* 
 
Normal number of bowel movements per day when UC is NOT active (transcribed from medical 
history): _______ 
 
0 – Normal number of stools for this subject 
1 – One to two stools more than normal 
2 – Three to four stools more than normal 
3 – Five or more stools more than normal 
 
* Each subject serves as his or her own control to establish normal stool frequency and the 
degree of abnormal stool frequency. 
 
2. Rectal Bleeding Subscore ** 
 
0 – No blood seen 
1 – Streaks of blood with stool less than half the time 
2 – Obvious blood with stool most of the time 
3 – Blood alone passed 
 
** The daily bleeding score represents the most severe bleeding of the day. 
 
3. Physician’s Global Assessment Subscore *** 
 
0 – Normal (subscores are 0) 
1 – Mild disease (subscores are mostly 1’s) 


 
 
 
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2 – Moderate disease (subscores are mostly 1 to 2) 
3 – Severe disease (subscores are mostly 2 to 3) 
 
*** The physician’s global assessment acknowledges the three other subscores, the subject’s 
daily record of abdominal discomfort and functional assessment, and other observations such as 
physical findings, and the subject’s performance status. 
 
4. Endoscopy Subscore: Findings of colonoscopy/flexible sigmoidoscopy 
 
0 – Normal or inactive disease 
1 – Mild disease (erythema, decreased vascular pattern, mild friability) 
2 – Moderate disease (marked erythema, absent vascular pattern, friability, erosions) 
3 – Severe disease (spontaneous bleeding, ulceration) 
 
 


 
 
 
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Complete applicable score for the visit. 
 
Partial MAYO Score 
 
_________  
+ 
_________  
+ 
_________ 
= 
_________________ 
Subscore 1  
 
Subscore 2  
 
Subscore 3 
 
Partial MAYO Score 
 
MAYO Score 
 
_________________  +  
_________  
=  
__________ 
Partial MAYO Score   
Subscore 4 
 
Mayo Score 
 
 


 
 
 
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19 
APPENDIX B:  COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS. 
 
The National Cancer Institute’s “Common Terminology Criteria for Adverse Events” (CTCAE) 
v4.03 is attached for reference.  CTCAE toxicities are graded on a scale of 0 to 4.  If a specific 
event is not included in the toxicity scale, the following scale should be used to grade the event: 
 
Grade 
Definition 
0 
No toxicity 
1 
Mild toxicity (usually transient, requiring no special treatment and 
generally not interfering with usual daily activities, e.g., colds) 
2 
Moderate toxicity (usually ameliorated by simple therapeutic 
manoeuvres, and impairs usual activities, e.g., pneumonia treated as 
an out-patient) 
3 
Severe toxicity (requires vigorous therapeutic intervention, interrupts 
usual activities.  Hospitalization may or may not be required, e.g., 
pneumonia requiring hospitalization or intravenous antibiotics) 
4 
Life-threatening toxicity (requires hospitalization, e.g., immediate risk 
of death from event and not reactions that had it occurred in a more 
serious form, might have caused death)