HLA-Identical Sibling Renal Transplant Tolerance With Donor Hematopoietic Stem Cells and Campath-1H
Phase: NA
Conditions: Immunosuppression, Kidney Transplantation, Graft Rejection
Sponsor: Northwestern University

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Synopsis 
 
 
Title:  
 
 
HLA-Identical Sibling Renal Transplant Tolerance With 
Donor Hematopoietic Stem Cells and Campath-1H 
 
Sponsor:  
 
National Institutes of Health (National Institute of Diabetes 
and Digestive and Kidney Diseases Branch) and the 
Comprehensive Transplant Center of Northwestern 
University 
  
Principal Investigator: 
Joseph Leventhal, MD, PhD 
 
Accrual Objectives:  
 30 recipients, 30 donors, 110 control subjects, and up to 60 
parents 
 
Study Design: 
 
To induce immunologic tolerance in non-randomized, kidney 
transplant recipients in a single center study. 
 
Study Duration:  
 
3 years, with clinical and laboratory routine follow-up for at 
least 10 years. 
 
Primary Study Objectives The primary objectives of this trial are: 
 
1.) To remove all immunosuppressive therapy from recipients of HLA-identical sibling renal 
transplants within 24 months of transplantation. 
2.) To detect and follow cellular (macro) chimerism of donor hematopoietic stem cell 
(DHSC) lineages and the generation of T-regulatory cells using specialized 
immunomonitoring assays for these donor/recipient pairs to demonstrate specific 
immunologic unresponsiveness. 
3.) To investigate the safety and efficacy of a treatment regimen consisting of induction 
therapy with Campath-1H and steroid-free low dose maintenance immunosuppression, 
consisting of mycophenolate mofetil (MMF)/Myfortic and tacrolimus converted to sirolimus.  
This is to be followed by complete withdrawal of immunosuppression beginning at one year, 
at a minimum, post-transplant, in recipients who have also been given four infusions of 
purified donor hematopoietic CD34+ stem cells (DHSC). 
 
Primary Outcomes 
The primary endpoints are:  
1.) Patient and graft survival measured at the one-year timepoint post-transplant. 
2.) The ability to withdraw immunosuppression as above 24 months post-transplant with 
follow-up to 10 years. 
 
Secondary Outcomes      Secondary safety and efficacy endpoints are as follows: 
1) 
Patient and graft survival measured at the three and five year time points post-
transplant. 

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2) 
Incidence rate of biopsy-proven acute rejection, defined as a renal biopsy 
demonstrating acute cellular or humoral rejection of Banff Grade IA or greater. 
3) 
Incidence of chronic allograft nephropathy, determined using renal biopsies and 
laboratory values, including 24 hour urine protein excretion. 
4) 
Incidence of graft versus host disease (GVHD). 
5) 
Incidence of adverse events associated with renal transplantation and 
immunosuppression, including infections, malignancies, post-transplant 
lymphoproliferative disease (PTLD), thromboembolic events, hyperlipidemia, 
leukopenia, thrombocytopenia, GI toxicity, and cytokine release. 
 
ELIGIBILITY 
 
Inclusion Criteria  
1. 
Patient has been fully informed, has signed a dated IRB-approved informed consent 
form obtained directly by the P.I., Co-P.I., sub-I and/or research nurse and is willing to 
follow study procedures for the duration of the study (for up to 10 years). 
2. 
Recipient must have a hematocrit of ≥ 33%, or 31% if patient is considered somewhat 
fluid overloaded. 
3.  
Age 18-65 years. 
4. 
Weight > 40 kg. 
5. 
Primary renal allograft: living related (HLA-identical donor-recipient sibling pairs) 
6. 
Negative B-cell and T-cell cross-match and (10%) PRA using cytotoxicity or flow 
cytometry. 
7. 
Women of childbearing potential will be required to have a negative qualitative serum 
pregnancy test. See also #11 below. 
8. 
Patients are to be studied equivalently as they become available for transplantation 
using these criteria, without regard to gender, race, or ethnicity. 
9. 
Normal echocardiogram with an ejection fraction >50%. 
10. Male participants with reproductive potential must agree to use approved methods of 
birth control during treatment with Campath-1H and for a minimum of 6 months 
following their last dose of Campath-1H.  Female participants of childbearing potential 
must agree to use approved methods of birth control for the duration of their 
participation in the study. 
11. Patient must agree to be followed every 2 months after year 3, for up to 10 years. 
 
Exclusion Criteria  
1. Patient has previously received or is receiving an organ transplant other than a kidney.  
2. Patient is receiving an ABO incompatible donor kidney.  
3. Recipient or donor is ELISA positive for human immunodeficiency virus (HIV), antibody 
positive for hepatitis C, or surface antigen positive for hepatitis B. 
4. Recipient or donor is positive for TB (or under treatment for suspected TB), or has had 
previous exposure to TB (positive Mantoux) who has not undergone an accepted course 
of treatment. 
5. Patient has a current malignancy or a history of malignancy (within the past 5 years), 
except non-metastatic basal or squamous cell carcinoma of the skin, or carcinoma in situ 
of the cervix that has been treated successfully.  

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6. Patient has uncontrolled concomitant infections and/or severe diarrhea, vomiting, active 
upper gastro-intestinal tract malabsorption or an active peptic ulcer or any other unstable 
medical condition that could interfere with study objectives.  
7. Patient is currently receiving an investigational drug or has received an investigational 
drug within 30 days prior to transplant.  
8. Patient is currently receiving any immunosuppressive agent.  
9. In the judgment of the investigator, it is anticipated that the patient will be unable to take 
medications orally or via nasogastric tube by the morning of the second day following 
completion of the transplant procedure. 
10. Concurrent use of warfarin, fluvastatin, astemizole, pimozide, cisapride, terfenadine, or 
ketoconazole.  
11. Patient has a known hypersensitivity to tacrolimus, Campath-1H, Thymoglobulin, 
sirolimus, MMF or corticosteroids.  
12. Patient is pregnant or lactating.  
13. Patients with a screening/baseline total white blood cell count <4000/mm3; platelet count 
<100,000/mm3.  
14. Patient is deemed unlikely to comply with the visits scheduled in the protocol. 
15. Patient has any form of substance abuse, psychiatric disorder or a condition that, in 
opinion of the investigator, may invalidate communication with the investigator.  
16. It is expected that tacrolimus cannot be instituted for longer than 5 days postoperatively.  
17. Patients with a cytotoxic PRA value >10% at any time prior to enrollment. 
18. Patients with Graves disease will be excluded unless previously adequately treated with 
radioiodine ablative therapy. 
19. EBV negative recipients of an EBV positive donor. 
20. History of Idiopathic Thrombocytopenic Purpura (ITP) or Thrombotic Thrombocytopenic 
Purpura (TTP) 
 
Glossary of Abbreviations 
 
ADCC 
Antibody dependent cell-mediated cytotoxicity 
ALG 
Anti-lymphocyte globulin (rabbit) 
ALT(SGPT)     
Alanine Aminotransferase 
APC 
Antigen-presenting cells 
AST(SGOT) 
Aspartate Aminotransferase 
ATG 
Antithymocyte Globulin 
ATGAM                    
(horse) Anti-thymocyte globulin induction therapy 
BMI 
Body Mass Index 
BSA 
Bovine serum albumin 
CAD 
Cadaver 
cDNA                      
Complementary DNA 
CIOMS                      
Council for International Organizations of Medical Sciences 
CML 
Cell-mediated lympholysis 

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CMV 
Cytomegalovirus 
CRF 
Case Report Form 
CRU 
CsA 
Clinical Research Unit 
Cyclosporine 
CTC 
Common Toxicity Criteria 
CTLp 
Cytotoxic T Lymphocyte Precursors 
D5W 
5% Dextrose and Water 
\
DBMC 
Donor-specific bone marrow cells 
DHSC 
Donor hematopoietic stem cells  
DSMB                     
   Data and Safety Monitoring Board 
DTH                           
Delayed Type Hypersensitivity 
EBV 
Epstein-Barr Virus 
ECG 
Electrocardiogram 
EDTA 
Ethylenedinitrilo Tetraacetic Acid 
ELISPOT           
Enzyme-linked Immunospot 
FDA                          
Food and Drug Administration 
GCP 
Good Clinical Practice 
GCSF                      
Filgrastim (Neupogen) 
GFR 
Glomerular Filtration Rate 
GGT 
Gammaglutamyltransferase 
GI 
Gastrointestinal 
GVHD                     
Graft vs. host disease 
HBSAg                    
Hepatitis B surface antigen 
HCG 
Human Chorionic Gonadotropin 
HDL 
High density lipoprotein 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
IV 
Intravenously 
ICH 
International Committee on Harmonization 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ITN 
Immune Tolerance Network 
ITT 
Intent-to-treat 
LDH 
Lactic Dehydrogenase 
LDL 
Low density lipoprotein 
LRD 
Living-related donor 
mAb 
Monoclonal antibody 

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MedDRA                  
Medical Dictionary for Regulatory Activities 
MGH 
Massachusetts General Hospital 
MLC 
Mixed lymphocyte culture 
MLDC                      
Mixed lymphocyte dendritic cell culture 
MMF 
Mycophenolate Mofetil 
mRNA                     
Messenger RNA 
NCI 
National Cancer Institute 
NIAID                     
National Institute of Allergy and Infectious Diseases 
NIDDK                     
National Institute of Diabetic, Digestive and Kidney Diseases 
NMFF                     
Northwestern Medical Faculty Foundation 
NMH                     
Northwestern Memorial Hospital 
NUIRB                  
Northwestern University Institutional Review Board 
NUCRU               
Northwestern University General Clinical Research UnitCenter 
PBL 
Peripheral blood leukocyte  
PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PCR 
Polymerase Chain Reaction 
PP 
Per protocol 
PRA 
Panel Reactive Antibody 
PTLD                       
Post-Transplant Lymphoproliferative Disease 
QID 
Four times a day 
RdD 
Recipient-derived donor 
RdR 
Recipient-derived recipient 
SAE 
Serious Adverse Event 
SAP 
Statistical analysis plan 
TB 
Tuberculosis 
TCD 
T-Cell Depletion 
TMP 
Trimethoprim 
TSH 
Thyroid-Stimulating Hormone 
 
 

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1 
Background Information and Scientific Rationale 
1.1 Background 
 
1) HLA-Identical renal transplant recipients of sibling donors have a ten-year actual (death-
uncensored) graft survival of 65-70% (Cecka JM, UCLA, National Transplant Registry, 
1993-1994 to 2003-2004). This 10-year data-point is on a slope that has a (late) downward 
curve, in all probability reflecting the cumulative effects of immunosuppressive therapy 
[since close to 20% mortality has occurred by 10 years with a median recipient age at 
transplant of 40±10.8 (S.D.) years], as well as chronic allograft nephropathy/rejection and (a 
few) disease recurrences.   
 
Even in this most recent era immunosuppressive therapy continues to be the main cause of 
early and late morbidity and mortality, in this most favorable group of recipients. These data 
contradict the ethical argument against immunosuppressive withdrawal “because such 
favorable matches do so well”. They override considerations supporting the continuous 
expense and the greater susceptibility to opportunistic infections, cancer, accelerated 
cardiovascular disease, diabetes, and blindness, as well as (in over 50% of recipients) the 
lack of rehabilitation to societal norms, so as to maintain long-term graft survival, which still 
results in chronic deterioration and loss of life. 
 
2) In preliminary observations, we are now following 7 renal transplant recipients of HLA-
identical sibling donors between 12 and 24 months post-operatively, who received the two-
dose Campath-1H induction regimen on (steroid-free) maintenance half-dose tacrolimus 
(Tacro) converted to sirolimus (Siro) at 2-3 months postoperatively together with between 
250 and 1,000 mg per day MMF. No rejections or other serious adverse events have 
occurred. 
 
3) This principal investigator has previously had and is still following patients in an ITN 
supported ongoing study (University of Miami Medical Center) based on living-related 1-
haplotype-matched renal transplant donor/recipient pairs undergoing one DHSC infusion 
peri-operatively, with a boost infusion three to four months postoperatively using Campath-
1H induction and (steroid-free) one-half dose Tacro with conversion to Siro (before the boost 
infusion), and one-half dose MMF maintenance conditioning before the withdrawal phase. 
Immunosuppression is being withdrawn over 30 months [beginning at 12 months 
postoperatively-Siro and at 24 months postoperatively-MMF]. Renal transplant biopsies are 
being performed at surgery and at 1, 2, and 3 years postoperatively to detect any evidence of 
cellular or humoral rejection. Such adverse findings would eliminate the recipient from the 
protocol and cause resumption of immunosuppressive maintenance (see current ITN protocol 
#022ST). Four patients have been transplanted and infused with DHSC and five controls 
have been transplanted who have been given the same immunosuppressive regimen without 
DHSC. (No drug withdrawal was performed in the controls.)  Clinical follow-up in the 
controls have demonstrated 2 of 5 with mild rejection episodes 20 month data on the first of 
these recipients shows ten-fold more chimerism than seen in our bone marrow infused kidney 
transplant recipients in previous protocols1,2 as well as (twofold) higher numbers of 
CD4+CD25+ T regs and Fox-P3 mRNA copies in CD3+ cells in peripheral blood.  For the 
more recent result update in the DHSC infused patients see Section 2.1.5. 
 

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4) Campath-1H is a humanized mAb directed to CD52 determinants on the surface of human 
leukocytes, of which there are an estimated 400,000 binding sites per cell. Waldmann found 
that optimal T-cell depletion was attained with the IgG1 isoform (1H).3 While the precise 
mechanisms of action are incompletely understood, it is apparent that this antibody prevents 
T-cell activation via CD45-signaling events and does not interfere with T-cell receptor 
activation, which might be highly relevant for future tolerance induction protocols. This 
treatment has proved to be highly effective in bone marrow transplantation for T-cell purging 
to eliminate graft versus host disease in over 2,000 participants treated in Europe in the 
treatment of B-cell lymphomas.3, 4, 5 Efficacy has recently been demonstrated in 
nonmyeloablative conditioning for stem cell therapy. Conditioning regimens based on 
Campath, cyclosporine, and donor-marrow stem cell therapy provided durable engraftment in 
62 of 64 recipients, with macrochimerism demonstrable in 31 cases.6, 7  The incidence of 
graft versus host disease (GVHD) was extremely low in this and other recent series, 
suggesting that Campath-1H treatment was protective against this complication.8 
This antibody has been particularly effective in control of autoimmune diseases, including 
acute vasculitides9, multiple sclerosis10, and in autoimmune cytopenias11, 12 
Although not FDA approved for renal transplantation, this agent has been used in an 
estimated 2000 renal transplant recipients (off label) over the last 10 years in the United 
States and elsewhere.  
 
5) It is a well-known phenomenon transcending phylogeny in several inbred experimental 
animal species enabling defined immune studies (fetal and adult inbred mice, rats, and pigs), 
that the lesser the immunogenetic disparity, the greater the feasibility of inducing donor-
specific immunological tolerance to transplanted organs and tissues. Mixed or total DHSC-
derived chimerism has been a basic component of the majority of these most robust tolerance 
states. There is also less likelihood of GVH reactions, the closer the donor/recipient match in 
these inbred strains. Both of these (tolerance and lack of GVH) have indeed proven to be the 
case in the myeloma human renal failure recipients of HLA-identical donor bone marrow and 
kidneys in the Massachusetts General Hospital series of Spitzer et al.13 
 
1.2 Hypothesis: 
It is our hypothesis that a four-dose (peri-operative and 3, 6, and 9-month boost) DHSC 
infusion protocol using two-dose Campath-1H induction combined with transient 
(conditioning) Tacro/Siro and MMF therapy will result in a high degree of macrochimerism 
(>10%), and a robust prolonged donor-specific (post-thymic) immunoregulatory condition 
that will allow renal transplant survival in the absence of permanent immunosuppression. 
 
Innovative mechanistic studies will include a novel HLA-identical chimeric PCR-Flow serial 
analysis based on non-HLA (cytokine gene) polymorphisms, other marker detection, split 
tolerance ELISPOT and micro-CML assays, in-vivo delayed type hypersensitivity (DTH), 
amplified HLA-identical mixed lymphocyte dendritic cell assays and molecular and flow 
cytometric analysis for T-cell and dendridic cell regulatory markers in peripheral blood and 
iliac crest marrow performed in our laboratories. These will be complemented with sensitive 
newer assays for donor-specific and non-specific (HLA and non-HLA) antibody studies in 
serial long-term follow-up. 

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1.3 Description of Investigational Product 
1.3.1 Donor Stem Cells 
This section briefly describes the collection, processing and delivery of the CD34+ enriched 
cells, and refers to the included SOPs and other appropriate documents in the Northwestern 
Memorial Hospital Cell Processing Center.  These SOPs, etc., are what will be used for every 
procedure step, and are included at the end of the entire protocol.   
These are numbered RWC 2.2.04, BC GEN 1.2.13, BC GEN 1.2.12, BMT 3.1.02, BMT 
3.1.03, BMT 3.1.10, BMT 4.1.01, BMT 5.1.02, BMT 10.1.01, HSCT 3.04 and also include a 
Table of Contents and  Product Certification of Analysis of Reagents, as well as Final 
Product Certificate of Analysis for Product.  Briefly, these stem cell procedures, as 
mentioned, are divided into three sections:  1.  Collection; 2.  Processing; 3.  Delivery.   
1.  Collection and 2.  Processing of Iliac crest bone marrow cells. 
Iliac crest bone marrow or peripheral blood progenitor cells (PBPC) will be harvested and/or 
processed for the following purposes: 
a. Donor BMCs to infuse into the recipient 
Approximately 750 mL of iliac crest bone marrow will be harvested under general 
anesthesia in a surgical suite from the donor after the donor transplant nephrectomy.  A 
bone marrow mononuclear “buffy coat” will be prepared using the GAMBRO Spectra 
apheresis instrument equipped with bone marrow buffy coat software set-up.  The 
allogeneic progenitor cells (buffy coat cells) will be further processed to isolate CD34+ 
progenitors using the Miltenyi CliniMACS system.  The isolated CD34+ cells are the 
investigational drug (cellular product) that will be tested in this protocol. 
 
MILTENYI CLINIMACS CD34+ CELL SELECTION 
Both sources of donor stem cells (intraoperative iliac crest bone marrow and HPC-A 
(leukapheresis) cells) will be positively selected for CD34+ cells prior to patient 
administration according to manufacturer’s instructions using the Miltenyi CliniMACS 
instrument.  Breifly, according to manufacturer instructions for operation of the CliniMACS 
device: 
 
1. HPC collection(s) will be washed with a phosphate buffered saline solution containing 
0.5 percent human serum albumin (HSA) and ethylene diamino-ethyl-tri-acetic acid 
(EDTA) to remove most of the platelets from the product and to reduce the product 
volume. 
 
2. Following the platelet wash step, the cells will be incubated with the CD34 monoclonal 
antibody-super-paramagnetic particle reagent supplied in the kit for 30 minutes and 
washed to remove unbound CD34 labeling agent. 
 
3. After cell labeling, the cells will be transferred to a sterile transfer pack, connected to the 
Miltenyi CliniMACS Selection device and an automated CD34 Selection program will be 
initiated.  The selection medium used throughout the procedure will be the 
manufacturer’s PBS/HSA/EDTA medium. 
 
4. At the completion of the selection process, an aliquot of the cells will be analyzed to 
determine the quantity and purity of the CD34+ cells and to assess the log depletion of 

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donor CD3+ T-lymphocytes.  The desired composition of the donor graft is: CD34+ cell 
dose > 2 x 106 CD34+ cells/kg.  T-lymphocyte cell dose < 5 x 104 CD3+ cells/kg. 
 
5. Specific details of the CD34 selection procedures can be found in the standard operating 
procedures (SOPs) located in Appendix II. 
 
b. Recipient BMCs for rescue 
 
Approximately 500 mL of iliac crest bone marrow will be harvested under general 
anesthesia in a surgical suite from the recipient prior to administration of alemtuzumab.  
This bone marrow will be minimally manipulated to reduce product volume only and 
will be frozen following standard cryopreservation methods for bone marrow.  This 
product will serve as a frozen “back-up” rescue source of autologous stem cells in the 
event that graft vs. host disease occurs in the recipient postoperatively to merit its use.  
c. Donor Mobilized HPCs 
Approximately 250 mL of peripheral blood progenitor cells (PBPCs) will be harvested 
from the donor following the harvest of the donor bone marrow cells.  The PBPCs will 
be collected using the GAMBRO Spectra apheresis instrument following standard 
HPC-A collection procedures currently in use at NMH Hematopoietic Stem Cell 
Transplantation Program.  Briefly, a standard volume leukapheresis (~ 12 L) will be 
performed on two successive days between two to three months postoperatively to 
obtain sufficient PBPCs  to permit isolation of peripheral blood CD34+ cells.  
(Mobilized by successive daily administration of GCSF).  The first day’s leukapheresis 
CD34+ product is to be administered to the recipient the same day (freshly) after the 
CliniMACS separation.  The second day’s leukapheresis product, after CliniMACS 
purification, will be divided into two equal aliquots to be administered after 
cryopreservation and thawing (at the bedside) at month 6 and 9 respectively.  The 
Miltenyi CliniMACS system is to be used for CD34+ isolation.  The isolated CD34+ 
cells are the investigational drug (cellular product) that will be tested in this protocol.   
 
3.  Delivery and Infusion of the Product 
These two methods are also described in the accompanying documents (#BMT 10.1.01 and 
HSCT 3.04) as mentioned at the end of the entire protocol consistent with methodology 
performed for stem cell patient delivery at Northwestern Memorial Hospital.  
In addition, a plan of action for all positive test results during the aforementioned procedures 
is included in these documents. Please refer to the QAP for a description of the notification 
of a positive test result following the QAP plan.  Adverse event reporting and corrective and 
preventive action plans will be used to notify the patient’s physician or study PI, investigate 
the incident and take appropriate corrective and preventive actions.  The incident (s) is to be 
reported to appropriate local, state or federal agencies as required.  This plan includes a 
mechanism to notify the PI in the event that a positive result is reported, in the event of 
positive microbial cultures, notification of manufacturing deviations, or in the event of post 
transfusion reactions.  Specifically, the PI is then to notify the Northwestern Institutional 
Review Board, the NIDDK sponsor, the Data Safety Monitoring Board and the FDA in an 
appropriate time sequence defined by the definitions of severe adverse reaction (included 
previously in this IND application). 
These products have been used 7 times in a NIAID, VA Merit Review, ITN and FDA 
approved study being conducted in 1 Haplotype matched, DHSC infused renal transplant 

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recipients now in progress in Miami.  They have also been used in many other stem cell 
therapies in hemato-oncology protocols at Northwestern, all without significant adverse 
events. 
 
2 
Summary of Preliminary Work 
Over the past decade, we have taken several steps towards understanding the mechanisms of 
specific immunological tolerance in a series of clinical and ex vivo studies in human kidney 
transplant recipients of DBMC infusions from CAD and LRD. Demographically similar non-
infused but non-randomized controls were given equivalent immunosuppression with OKT3 
or daclizumab antibody induction and tacrolimus, MMF, and methylprednisolone 
maintenance. Follow-up included ex vivo assays of chimeric cell quantitation and immuno-
regulatory function. We also described in vitro assays of the general immunoregulatory effect 
of bone marrow non-T and T cell subsets, stemming from the surprising finding, after bone 
marrow infusion, of the significant increase in chimeric (donor) cells in the sequestered en-
vironment of the recipient marrow compartment, as opposed to peripheral blood.  The fol-
lowing summaries therefore also include these published studies, as well as our initial 
Campath-1H (unpublished) work within the past year. 
 
2.1 Summary of Clinical Experience 
2.1.1 Campath-1H and Cyclosporine in Renal Transplantation 
Watson, CJ 14 used Campath-1H for induction prophylaxis in 31 participants undergoing 
renal transplantation. Campath-1H was administered at a dose of 20 mg on Day 0 and 
repeated on Day 1, and after a 72-hour delay cyclosporin (target trough level 75 to 125 
ng/mL) was initiated without glucocorticoids and without any other maintenance drug 
therapy.  At a follow-up of five years, there has been a 22.6% incidence of acute rejection 
(16.1% at 1 year with 79% graft survival at five years and 88% participant survival at five 
years) Watson, CJ, Calne R, et al. Am J Transplant 2005;5:1347-53.  There was prolonged 
and profound depletion of circulating lymphocytes with a mean lymphocyte count at 1-year 
follow-up of approximately 800/mm.  Despite this low lymphocyte count, there has not been 
an increased incidence of infection or malignancy compared to conventional therapy.  In this 
series, Campath-1H did not prevent recurrent disease, 2 participants have had recurrent IgA 
nephropathy, and 1 participant had recurrent membranous glomerulonephritis.  This 
observation is similar to conventional therapy. 
 
2.1.2 Campath-1H and Sirolimus in Renal Transplantation 
Data on the use of Campath-1H in renal transplantation were reported by Kirk et al.15  This 
trial was designed to determine whether pretransplant administration of Campath-1H would 
allow allotransplantation without maintenance immunosuppression. Eight living related-
donor renal-allograft recipients were given 3 doses of Campath-1H (0.3 mg/kg), initiated 
before transplantation. There was rapid clearance of T cells from circulation, which persisted 
beyond 3 months, and up to 12 months in some cases. Normal renal function was observed in 
all participants at 2 weeks after transplant. Graft histology was normal at 7 days.  By 3 to 4 
weeks, all participants had some degree of renal dysfunction, which improved following the 
initiation of sirolimus.  Preliminary data from this study has demonstrated that Campath-1H 
provided a 2-week window of decreased immune responsiveness.  More recent similar 
steroid avoiding protocols have been described in LRD and CAD renal transplant recipient 

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by Knechtle, et al. using a two-dose Campath-1H induction regimen and continuous 
sirolimus therapy.16 
 
2.1.3 Campath-1H, Tacrolimus and MMF in Renal Transplantation 
At Northwestern University/Northwestern Memorial Hospital, a single intraoperative dose of 
30 mg Campath-1H was used in combination with tacrolimus (trough levels of 4-6 ng/mL), 
and MMF (1 gm daily), beginning after September 2001.  In May 2002, 23 consecutive 
kidney transplant recipients were reported without rejection and all with good function.  One 
participant had a urinary tract infection. Campath-1H was associated with prolonged 
lymphophenia lasting 4 to 6 months.17   Kaufman et al recently reported on Northwestern’s 
single center experience with Campath 1H induction in 123 renal transplant recipients 18. 
Again a single intraopertive dose of Campath 1H was used with prednisone-free maintenance 
immunosuppression consisting of tacrolimus and MMF.  One year acute rejection rate was 
15.4%. Patient and graft survival at 3 years post-transplant were 95.9 and 91.9 %, 
respectively.  Infectious complications in Campath 1H patients were not qualitatively 
different from an historical cohort of patients receiving a non lymphocyte depleting induction 
agent (Simulect).  Currently, over 1000 renal transplant recipients have been treated with 
Campath 1H induction at Northwestern. 
 
2.1.4 Campath-1H Experience in Transplantation at the University of Miami 
 
The use of Campath-1H as induction therapy in renal transplantation: preliminary 
results.19 In attempt to reduce both initial and long-term (nephrotoxic) calcineurin inhibitor 
maintenance dosage and totally eliminate maintenance corticosteroids.  In Miami, the PI used 
Alemtuzumab (Campath-1H) as induction therapy in first cadaver and non-HLA identical 
living donor renal transplantation.  Forty-four de novo renal allograft recipients were treated 
with Campath-1H (0.3 mg/kg) on day 0 and day 4 postoperatively preceded by 
methylprednisolone boluses. Maintenance target 12-hour Tacrolimus trough levels of 5-7 
ng/ml were operational from the outset as well as (reduced) mycophenolate mofetil dosage of 
500 mg twice daily.  No corticosteroids were planned to be given after the first week 
postoperatively.  With a median follow-up of 9 (range 1-19) months, patient and graft 
survival were each at 100%.  Biopsy-proven acute rejection was diagnosed in 4 patients.  A 
total of 4 patients developed infections that required hospitalization.  Thirty-eight recipients 
remain without the need for long-term corticosteroid therapy.  In an early assessment, the 
combination of Campath-1H, low dosing of tacrolimus and mycophenolate mofetil, and 
avoidance of maintenance corticosteroid use appears to be safe and effective for kidney 
transplant recipients.  Long-term outcomes will be reported in the future. 
 
A randomized trial of three renal transplant induction antibodies: early comparison of 
tacrolimus, MMF and steroid dosing, and newer immune-monitoring by the PI.19,20  In a 
randomized trial using three different antibody induction agents in 90 first renal transplant 
recipients from cadaver donors, Group A received Thymoglobulin, Group B received 
Alemtuzumab, and Group C received Daclizumab.  Maintenance immunosuppression 
included tacrolimus and mycophenolate in all three arms, and methylprednisolone in Groups 
A and C only (standard clinical institutional practice).  The targeted trough level of 
tacrolimus was between 8 and 10 ng/ml for Groups A and C, respectively, with a targeted 
mycophenolate dose of 1 gm twice daily.  However, in Group B, target tacrolimus trough 

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level was 4-7 ng/ml to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of 
mycophenolate, without steroid maintenance. In this 15-month median postoperative interval 
report, there were no notable differences in demographics and patient and graft survival.  
Acute rejection rates at one year were equivalent, i.e., 5/30 in all three groups (~16.6%).  In 
Group B, there was slightly lower renal function at one month, but no difference at one year.  
There was also significantly more leukopenia, but a greater percentage of T regulatory cells 
and number of Fox-P3 mRNA copies respectively, by flow cytometry and semi-quantitative 
PCR analysis in Group B.  This preliminary analysis has indicated that in Group B 80% of 
these patients remained steroid-free at 1-year postoperatively, with lower tacrolimus trough 
levels, and no difference in other adverse events. 
 
Campath-1H does not alter bone marrow cell regulatory function.20,21  We have 
previously reported in laboratory volunteers (in vitro) and renal transplant recipients (ex vivo), 
that bone marrow cells (BMC) are potent down-regulators of the immune response.  Also, the 
use of alemtuzumab (Campath-1H, C1H) for immunodepletion is associated with the most 
potent lasting effects yet seen on T cell immunity in renal transplantation. We questioned if 
the administration of C1H to kidney allograft recipients of donor bone marrow cell (DBMC) 
infusions would lead to stronger or weaker immunoregulatory effects.   In vitro Human BMC 
depleted of T cells (nT-BMC) were either untreated or treated with C1H and rabbit 
complement, and compared for their ability to down-regulate autologous or allogeneic T cell 
responses, and to generate T regulatory (T reg) cells.  The proliferative responses to anti-CD3 
monoclonal antibody of T cells derived from co-cultures with C1H-treated or untreated 
autologous nT-BMC were equally suppressed, i.e., an equivalent alteration in CD3 complex 
signaling, not regained by the addition of IL-2.  ATP levels were also markedly reduced in T 
cells both from C1H-treated and untreated nT-BMC co-cultures. The ability of C1H-treated or 
untreated nT-BMC to suppress autologous T cell cytotoxic function was also equivalent, with 
a marked, but equivalent, capacity to induce CD4/CD25high T regs from CD3+ cells, which 
effectively down-regulated cytotoxic T cells.  To mimic the clinical infusion of DBMC into 
(allogeneic) recipients, PBL were also cultured with allogeneic C1H-treated and untreated nT-
BMC.  T cells derived from these cultures secondarily stimulated with the same donor mature 
APC, showed suppressed cytotoxicity by 85% and 54%, respectively.  These in vitro studies 
suggest that C1H does not abrogate BMC immunoregulation and thus may allow its lympho-
depleting effect to be synergistic. 
 
2.1.5 DBMC Experience in Transplantation  
In the PI’s experience at the University of Miami between September 1994 and May 1998, 
63 CAD renal transplant recipients of either one or two postoperative vertebral DBMC 
infusions were prospectively compared with 219 demographically equivalent (non-
randomized) non-infused controls given equivalent OKT3, tacrolimus, MMF, and 
methylprednisolone immunosuppression. There was at least a 1 HLA DR antigen match pres-
ent between donors and recipients in all cases.  Clinical follow-up ranged from 2.9-6.3 years 
(mean, 4.7 years).  Only 2/63 (3.2%) DBMC recipients had biopsy-proven chronic rejection, 
whereas 41/219 (18.7%) showed chronic rejection in the controls (p = <0.01).  If death with a 
functioning graft was excluded, graft survival was 94.1% in the DBMC group and 79.8% in 
the controls (p=0.039). Forty controls had deteriorating renal function compared with 2 in the 
DBMC group (p=0.04). In the DBMC group, chimerism in iliac crest marrow aspirates had 
increased in sequential yearly PCR-Flow measurements between 1 and 4 years 

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postoperatively, to average 1.3±0.36% (S.E.), but not in the controls.1  More recent follow-up 
at 10 years has confirmed these findings with FoxP3 mRNA copies in the total T cell PBL 
compartment being five times that of the non-infused controls.(Cirocco et al, Transplantation 
2007, in press) 
 
Between November 1996 and May 2000, 47 LRD kidney transplants who received iliac crest 
DBMC in a single infusion 4 days postoperatively were (non-randomly) concomitantly com-
pared with 39 demographically similar non-infused controls. All were given either OKT3 
(n=26) or daclizumab (n=21) induction with maintenance tacrolimus, MMF, and 
methylprednisolone immunosuppression. Clinical follow-up ranged from 19.0-61.6 months 
(mean: 33.2 months). The incidence of acute rejection did not differ between groups.  
Immunosuppressive dosaging was somewhat (but not statistically) lower in the DBMC 
group.  Four-year actuarial patient and graft survival for the DBMC-infused group was 98% 
and 98%, and 98% and 95% in the controls (p=N.S.). DBMC chimerism by PCR-Flow in 
recipient iliac crest marrow increased more rapidly than might have been predicted from the 
CAD group, despite four times fewer DBMC infused. 2 
 
Preliminary Results of ITN Protocol (#022ST) Pilot Study: Four patients are being 
followed in a protocol of 1-haplotype matched LRD Campath-1H treated donor stem cell 
infused kidney transplant recipients with the intent to wean immunosuppression over a three 
year period starting one year post-operatively. 
 
In the first patient, R.O., 1.2 x 106 CD34+ donor iliac crest bone marrow-derived purified 
stem cells were infused five days post-operatively, one day after the second Campath-1H 
treatment. 
 
At 12 weeks, 2 weeks after conversion to sirolimus from tacrolimus 7 x 106  donor CD3+ 
cells were again infused after five days of Filgastrin CD34+ mobilization, from a peripheral 
blood leukapheresis product (<.05% T cell contamination). 
 
Table 1 shows the subsets of donor chimeric cells in recipient PBL and iliac crest marrow, 
compared with those seen at similar intervals in previous DBMC-infused recipients from 
series generated over the past 7 years (>10-fold higher than previously at similar points in 
time).   
Table 1. 

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Patient and Graft Outcome:  As a brief summation of that study, similar findings have been 
present in three other stem cell-infused patients—now all over one-year post-transplant, but 
one (DHSC Patient  #2, a 20 year old Latin female; RO) has had a rapid recurrence of focal 
segmental glomerulosclerosis (FSGS) that occurred  in the native kidney.  Although she is 
not yet on dialysis, she is nearing this point with a serum creatinine of  >5.0 mg/dl. 
 
DHSC patient #1, (a 28-year old Latin female) has a serum creatinine of 1.8 mg/dl, due to a 
recurrence of Type I membranoproliferative glomerulonephritis (MPGN) in the transplant, 
which has been very slowly progressive but in which allograft rejection has not been present 
on biopsy. (now 3 year post-transplant). 
 
Patient #3, (a 30-year old Latin male) is now two years after transplantation with a serum 
creatinine of 1.3 mg/dl.  He is about to be totally withdrawn from sirolimus (now on .5 
mg/day) to convert solely to MMF.   
 
Patient #4, (a 62-year old Caucasian male) is now 15 months post transplant with a serum 
creatinine of 1.4 mg/dl and is being slowly withdrawn from sirolimus, with MMF to remain 
for the additional several months after sirolimus withdrawal as per the ITN protocol.   
 
Patients #3 and #4 have also had no evidence of acute or chronic rejection clinically or on 
biopsy, but Patient #3 is very susceptible to dehydration, with having had some proteinurea 
attributed to sirolimus therapy, which has subsided as the drug has been slowly withdrawn. 
 
Treatment Received (Brief Summary):  Patients #1, #3 and #4 all received the DHSC 
infusions and planned immunosuppression of the protocol described in Section 1.1- i.e., two 
doses of Campath and half dosing of tacrolimus switched to sirolimus.  However, Patient #1, 
because of her recurrence of MPGN, has not had immunosuppression withdrawn and actually 
has had 4 mg of methylprednisolone added to her therapy.          
 
LRD Recipients a
Patient "R.O."
1.8x108±1.9x108 cells/kg in
one infusion perioperatively
Infused with 1.6x106  DHSC/kg
perioperatively and
7x106 DHSC/kg boost
3 months postoperatively
Comparison at 1 year post-transplant of peripheral blood PCR-Flow chimerism
of patient "R.O." given DHSC vs. the group of LRD recipients (previous series)
given ~50-fold more donor iliac crest whole marrow cells.
Time
1 year
1 year
N
33
1
Peripheral Blood
.18 ± .08
3.95
%b Donor CD3+ Cells
.10 ± .05
9.81
a
b A mean of 1.8 x 108 cells/kg infused ± 1.9 x 108 S.D. in the entire group.
Percent of the total (recipient and donor) CD3+ or CD34+ subset counted.
%b Donor CD34+ Cells

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As mentioned, Patient #2, with rapid recurrence of FSGS, has been on a modified dose of 
MMF and corticoid steroids as well.  She did not receive the second DHSC infusion because 
FSGS recurred before month 2.   
 
Patients #3 and #4 are on the withdrawal protocol as described.   
 
Chimerism:  Patients #1, #3 and #4 have chimerism levels hovering around 1% in iliac crest 
bone marrow aspirates by PCR-flow analysis, all performed within the last 3 months.   
 
2.2 Ex vivo and in vitro Correlates of Allogeneic and Autologous Immunoregulatory 
Mechanisms Evoked by Bone Marrow Cells (ex vivo = Cells Derived from the Chimeric 
Recipient; in vitro = Non-chimeric Bone Marrow Cells of Non-transplanted Individuals)  
 
Listed are titles of publications by the P.I. and colleagues (in citations section by number) 
that apply both to the clinical and mechanistic rationale from the present protocol. 
 
- Donor and recipient chimeric cells present in DBMC-infused renal transplant recipients: 
potent ex vivo regulators of recipient anti-donor immune responses.21 
- In vitro immunogenicity of cadaver donor bone marrow cells.22 
- Involvement of multiple alloimmune regulatory subpopulations of DBMC.23 
- Regulation of alloimmune (GVHD) reactions in vitro by autologous DBMC.24 
- DBMC inhibit the generation of autologous EBV-specific CTL, promote TH2 polarization 
and can cause the transfer of an anergic state between T cells of peripheral blood.25-28 
- Allogeneic DBMC induce in vitro "suppressor T cells" of autologous B cells.28 
- A novel CML micro-assay for the evaluation of regulatory cells.29 
- The human bone marrow as an immunoregulatory organ and the immunoregulatory role of 
chimerism in clinical organ transplantation.30-32  
 - Induction of auto-reactive regulatory T cells by stimulation with immature autologous 
dendritic cells.50 
- Killer cell immunoglobulin-like receptor polymorphisms in HLA-identical kidney 
transplant recipients: lack of 2DL2 and 2DS2 may be associated with poor graft function.51   
- A Novel Approach to Detect Donor/Recipent Immune Responses Between HLA-Identical 
Pairs.52 
 
2.3 Known and Potential Risks and Benefits to Human Subjects 
2.3.1 Potential Benefits 
Renal transplantation using Campath-1H induction combined with DHSC infusions from the 
same HLA identical sibling donor has the potential to allow acceptance of a donor renal 
allograft without chronic immunosuppression.  This treatment is believed to cause a greater 
degree of tolerance of the transplanted kidney by the body, thus, avoiding rejection and 
perhaps allowing the partial or even total withdrawal of the need for continuous use of anti-
rejection drugs.  This therapy has been undertaken in an attempt to offer renal transplantation 
and hence freedom from dialysis without the complications of long-term immunosuppressive 
drug administration.  Long-term complications of continuous immunosuppression   can 
include cataracts, osteoporosis, diabetes, atherosclerosis, hypertension, malignancy, plus the 
risk of chronic allograft rejection/nephropathy. 

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2.3.2 Potential Risks 
Participation in this study is associated with certain risks for both the recipient and the donor.  
These risks are summarized below. 
In general, patients with a transplanted organ are treated with immunosuppressive drugs that 
suppress the immune system.  These drugs produce additional adverse events occasionally 
including over-immunosuppression.  This may cause infections, including those caused by 
viruses, bacteria, and fungi, and by organisms that only produce disease in patients who are 
immunosuppressed.  Over-immunosuppression may also increase the risk for the 
development of cancer.  It is not known if infusion of donor bone marrow will result in an 
increase or decrease or similar risk of infection or cancers compared to standard therapy.  
   
2.3.2.1 Graft Versus Host Disease 
GVHD (graft versus host disease) although considered to be unlikely might occur, with the 
possibility of the donor bone marrow cells reacting against the recipient.  This has been seen 
in patients who have received bone marrow transplants to treat other diseases, such as 
leukemia, in which there has been an ablative procedure to totally knock out the recipient’s 
own marrow.    However, in this protocol, the patients are not to receive the marrow 
destroying therapy, which is probably a prerequisite for the development of graft vs. host 
disease.  Nonetheless, one unit of the recipient’s own marrow and recipient’s white cells will 
be stored before transplantation to treat GVHD.   
T-cell depletion (TCD) of the infused marrow derived cells, a strategy to decrease GVHD, is 
under investigation in stem cell transplantation for hematologic, oncologic and 
immunodeficiency diseases.  This technique effectively decreases the incidence and severity 
of acute GVHD, but with increased problems with graft rejection33-37 and relapse. Methods of 
graft manipulation may be based upon removing T lymphocytes (TCD) by a variety of 
antibody mediated approaches or physical methods of separation. Most recently, positively 
selected CD34+ progenitor cells are commonly used. Transplant related mortality outcome 
appeared to be predominantly affected by increased GVHD in recipients receiving peripheral 
blood cell grafts containing more than 2 x 105 CD3+ T cells/kg.  In patients receiving more 
than 2 x105 CD3+ cells/kg, the incidence of Grade II-IV GVHD was 55%. It is important to 
note that in all these studies, patients receive myeloablative regimens.  Perhaps the largest 
experience of TCD transplants occurring without preparative chemotherapy is in the setting 
of severe combined immune deficiency. In this disease, children often receive TCD 
haploidentical parental marrow. In most cases, GVHD that developed after the administration 
of TCD depleted marrow was mild (grade I or II) and required no treatment.  
 
2.3.2.2 Campath-1H 
Infusion-related events have been associated with the use of Campath. These events include 
rigors, fever, nausea/vomiting, fatigue, hypotension, shortness of breath, bronchospasm, 
chills, pruritis, headache, diarrhea, and urticaria and/or rash. In patients using Campath for 
oncology indications, Campath has been found to induce profound lymphopenia in patients 
given many-fold higher dosing than in renal transplant recipients in this study.  Campath 
therapy has been associated with the opportunistic viral, bacterial, and fungal infections, 
some of which were fatal.  Leukemia and lymphoma patients receiving Campath have 
experienced severe, prolonged, and in rare instances fatal myelosuppression.  Bone marrow 
aplasia and hypoplasia have been observed, as well as severe and fatal autoimmune anemia 
and thrombocytopenia in patients with chronic lymphocytic leukemia.38 

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Although Campath-1H therapy may lead to prolonged suppression of CD4+ cells, this has 
not been found to be associated with clinical infection in humans who have suppressed CD4+ 
cells following lymphoid irradiation or bone marrow transplantation.  Research participants 
in the Campath-1H trial in England (31 subjects) have not experienced any serious or life 
threatening infections following renal transplantation. Two subjects experienced 
opportunistic infections (systemic cytomegalovirus infection and an abdominal abscess 
attributed to reactivation of a prior tuberculosis infection) that responded to therapy.39 
Recent reports of an increased risk of Graves disease have only been seen in a single trial of 
participants with multiple sclerosis, where 1/3 of participants developed the disease 
following Campath-1H treatment in much higher dosing than used in the present study.  At 
Northwestern University we have not observed any cases of Graves disease in patients 
receiving Campath 1H as induction for solid organ transplantation. 
 
Patients with Graves disease will be excluded unless previously adequately treated with 
radioiodine ablative therapy.  This trial was unique in that very high doses of Campath-1H 
were given (100 mg given over 5 days) and Graves disease did not occur until 6 to 18 months 
later.  It has been presumed that this complication may reflect a genetic predisposition in 
participants with multiple sclerosis, since in controlled trials the complication has not been 
observed in other participants with chronic leukemias or in participants with vasculitides. 
Thyroid function tests will be monitored in the current trial.  
 
No long-term animal studies have been performed to establish the carcinogenic or mutagenic 
potential of Campath-1H or to determine its effects on fertility rates in male or female adults.  
Campath-1H may cross the placental barrier and cause significant fetal T and B lymphocyte 
depletion.  Male participants with reproductive potential must use approved methods of birth 
control during treatment with Campath-1H and for a minimum of 6 months following their 
last dose of Campath-1H. Female participants of childbearing potential must use approved 
methods of birth control for the duration of their participation in the study.  
 
In some oncology subjects treated with Campath, profound and long-lasting lymphopenia 
(decreased CD4+ and CD8+ lymphocyte counts) was observed, and total lymphocyte counts 
did not return to baseline levels even after 1 year post-therapy.  CBC and platelet counts will 
be monitored frequently if worsening anemia, thrombocytopenia, lymphocytopenia, or 
neutropenia is observed on study. 
 
2.3.2.3 Tacrolimus  
Tacrolimus administration will be based on trough blood levels (AM) to provide levels of 
between 4 and 7 ng/ml during its administration course.  These levels will be obtained daily 
as inpatients and with each clinic visit as outpatients (see calendar).  The tacrolimus dose 
should be decreased in the presence of adverse events as clinically warranted.   Risks 
associated with the administration of tacrolimus include hypertension, hyperkalemia, 
nephrotoxicity, neurotoxicity, posttransplant insulin-independent diabetes mellitus, 
myocardial hypertrophy (in most cases reversible upon dose reduction), and increased risk of 
renal insufficiency in patients with hepatic impairment. 
2.3.2.4 Sirolimus  
Sirolimus dosage, when instituted (2 to 3 months postoperatively) will be based on AM 
trough levels of 8 to 12 ng/ml.  These levels will be obtained with each clinic visit (see 

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calendar).  The sirolimus dose should be decreased in the presence of adverse events as 
clinically warranted. Risks associated with the administration of sirolimus include 
hypertriglyceridemia, hyperlipidemia, leukopenia, thrombocytopenia, anemia, neutropenia, 
nephrotoxicity, hypertension, anemia, nausea, vomiting, diarrhea, elevated liver enzymes, 
rash and acne. Sirolimus’ effect on the developing fetus is not known and is not 
recommended for administration to nursing mothers.  It is recommended that women of 
childbearing potential use effective contraception before, during and for at least 4 months 
following Sirolimus administration. 
 
2.3.2.5 Mycophenolate Mofetil (MMF) 
The MMF dose should be decreased in the presence of adverse events as clinically 
warranted. Risks associated with the administration of MMF include leukopenia, 
thrombocytopenia, anemia, neutropenia, gastritis, diarrhea, nausea, vomiting or dyspepsia. 
MMF’s effect on the developing fetus is not known and is not recommended for 
administration to nursing mothers.  It is recommended that women of childbearing potential 
use effective contraception before, during and for at least 4 months following MMF 
administration. 
 
2.3.2.6 Combination Therapy: DBMC, Campath-1H, Tacrolimus, Sirolimus, and 
MMF 
Other than the experiences cited 40 above, no information to date is available on the 
combination of DHSC and Campath-1H given together with either tacrolimus, sirolimus, or 
MMF.  Thus, at this time the risk profile of the combination of these therapies are not known.  
Potential risks may include increases in the incidence or severity of adverse events currently 
experienced when these agents are administered, in addition to other adverse events that are 
unforeseeable at this time.  Also, as with any immunosuppressive regimen, this combination 
may result in increased susceptibility to infection and the development of lymphoma and 
other malignancies. 
 
2.3.2.7 Other Risks   
The subject may experience some pain and bleeding at the site of the kidney transplant 
biopsy, discomfort, and/or blood in the urine following the biopsy. Additional risks of biopsy 
include bleeding in or around the kidney that can lead to a fall in blood pressure and rise in 
heart rate.  In the event that significant bleeding is suspected, either through clinical 
observation or a significant decrease in hemoglobin, a blood transfusion will be administered 
as necessary or the patient will be taken to the operating room for direct control of bleeding. 
 
3 
Objectives 
3.1 Primary Objectives 
The primary objective of this trial is to investigate the safety and efficacy of a treatment 
regimen consisting of induction therapy with Campath-1H and reduced dose maintenance 
immunosuppression (consisting of  MMF, Tacrolimus, and Sirolimus), followed by complete 
withdrawal of immunosuppression within 30 months of transplantation, in recipients of 
living-related (HLA identical) donor kidney transplants who have also been given four 
infusions of purified CD34+ DHSC. 
 

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3.2 
Secondary Objectives  
To assess the utility of various assays and biopsy information as putative tolerance markers 
in these kidney transplant recipients: 
• To use the PCR-Flow assay (modified for cytokine gene polymorphism) and VNTR 
assays both in recipient peripheral blood on a monthly basis and in iliac crest marrow at 3 
months, 6 months and yearly, in order to measure donor cell multi-lineage chimerism; 
• To use the newly developed cell-mediated lympholysis (CML) microassay, enzyme-
linked immunospot (ELISPOT) assay, and MLDC and in vivo DTH assays to 
demonstrate sequentially increasing donor and recipient chimeric marrow and peripheral 
blood cell immunohematologic subset regulatory effects; 
• To study lymphoid cells derived from yearly kidney biopsies (i.e., before and after 
planned withdrawal of immunosuppression); 
• To use limiting dilution analysis to detect partial or total clonal deletion. 
• To follow sensitive alloantibody assays;  
 
4 
Study Design 
4.1 Donor and Recipient Preparative Regimens 
Twenty recipients will be HLA identically matched with their living related donors. There 
will be occasions in which it will be determined that the standard tissue typing tests may not 
be enough to define HLA genotypic identity.  If this is the case more discriminatory tissue 
typing will be performed that may include tissue typing of both parents. One month before 
transplantation (at the time of consent), 1x109 recipient mononuclear cells are to be removed 
by a 7-liter leukophoresis and cryopreserved in aliquots. Fresh samples of donor and 
recipient PBMC are to be tested as responding, stimulating, and regulatory cells in MLC and 
CML reactions at this time. Leukophoresis (Cobe Spectra), without causing recipient anemia, 
will furnish these (non-regulatory) cryopreserved recipient cells for later comparison. 
 
4.2 
Initial Recipient Protocol and Immunosuppression (at and immediately post-
surgery) 
In the operating room, with induction of general anesthesia, 500 ml of iliac crest bone mar-
row will be removed from the recipient (inclusion criterion – hematocrit >33), cryopreserved 
for GVHD rescue (unlikely) and tested by PCR-Flow (baseline absence of chimerism).  
Some aliquots will be cryopreserved (from 100 ml of this marrow) for comparison in the 
mechanistic studies.  Then 250 mg of hydrocortisone and 50 mg Benadryl® will be 
administered intravenously (I.V.), followed by an infusion of 0.3 mg/kg of Campath-1H over 
2 hours, which will be completed about the time the clamps are released on the renal vascular 
anastomoses. At clamp release: 500 mg of methylprednisolone is administered. MMF (500  
mg) is infused I.V. at surgery and continued enterally at a dose of 500 mg/12 hr (half the 
usual dose).  Tacrolimus is instituted at 0.05-0.1 mg/kg every 12 hr enterally, when good 
renal function is established (serum creatinine decreases to <4 mg/dl), anticipated to be 
within 48 hr of surgery, targeting for A.M. trough levels of 5-7 ng/ml. 
4.3 
Donor Marrow Donation 
After the uncomplicated donor nephrectomy is completed, before anesthesia is withdrawn, 
750 ml of iliac crest marrow will be removed for CliniMACS stem cell purification, 
cryopreservation, and infusion 5 days later into the recipient (see below). 
 

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4.4 
Post-operative Campath-1H 
The second infusion of Campath-1H is to be given on post-operative day 4, also at a dose of 
0.3 mg/kg.  A change in the Campath 1-H dosage is allowed upon the Principial 
Investigator’s judgment to meet specific clinical needs of subjects.  This is preceded by 500 
mg of hydrocortisone I.V., 50 mg Benadryl® and 650 mg (2 tabs) Tylenol® by mouth to 
avoid any potential adverse reaction with the agent (package insert).  Using this regimen, a 
Campath-1H functional half-life of ~12 days is anticipated, with prolonged effects up to 1 
year.  No additional corticosteroids are to be administered. 
 
4.5 
Long-term Immunosuppression 
At 1 month post-operatively (assuming a normal rejection-free clinical course), tacrolimus 
will be withdrawn over 2 weeks, overlapping with the prior initiation of sirolimus in doses to 
achieve target trough levels of 8-15 ng/ml. (usually between 2-5 mg/day), MMF 500 mg 
twice daily will be maintained as tolerated. 
Note:  None of these agents are considered investigational.  Although Campath-1H is used 
clinically in organ transplantation (over 2000 patients treated in the United States in the past 
five years), it is not used routinely for this purpose, but has been indicated to date in the 
treatment of hematologic malignancies.  The decrease in dosing of Tacrolimus and MMF 
accompanying Campath-1H usage has been routine for those centers using Campath-1H in 
renal transplantation, as well as the absence of corticosteroids maintenance therapy (over 
1000 patients in the United States with more than 1 year of follow-up). 
 
4.6 
Donor Stem Cell Infusions (Please see section earlier in the protocol entitled           
Description of Investigational Product Section 1.3.1) 
4.6.1 First Infusion 
The first donor purified stem cells will be given on post-operative day 5 extracted from the 
iliac crest marrow aspirated at donor nephrectomy. After a 10 ml aliquot is taken, together 
with PBL for PCR-Flow analysis, the marrow will be purified by the CliniMACS® (CD34+ 
selection) 300i System.  From our previous experience in the Diabetes Research Institute at 
the University of Miami and in this NU/NMH stem cell processing unit in other protocols, 
approximately 1-2 x 106 cells/kg body weight can be expected as an infusion dosage with a 
CD34+ cell purity of ~75%, with ~0.5% T cell contamination. The cells will be cryo-
preserved, thawed, and administered at the recipient bedside (as in previous studies). 
 
4.6.2 Second, Third, and Fourth Infusion 
The second infusion of donor peripheral blood-mobilized stem cells, purified similarly, will 
be administered to the recipient (as a patient in the CRU for <12 hours) 2 to 4 weeks after the 
initiation of Sirolimus therapy (between 2 to 3 months postoperatively), once sirolimus 
trough levels have reached 8 ng/ml.  
Donors as outpatients will undergo standard 10-12 liter pheresis, at this time using peripheral 
access whenever possible. Donors will be administered G-CSF (filgrastim) subcutaneously 
for four consecutive days prior to pheresis with dose according to body weight (10 mcg/kg). 
A CD34+ cell assay of the product after CliniMACS® selection will be carried out.  The 
donors will then receive one more dose of G-CSF and undergo a second pheresis the 
following day, and CD34 selection performed. The plan is to administer at least 2x106 
cells/kg recipient body weight freshly from the first pheresis, after premedication with 
Benadryl® and hydrocortisone, but without additional Campath-1H treatment. 

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The product of the second pheresis will be cryopreserved and two aliquots successively 
thawed and infused into the recipients as above at 6 months and 9 months postoperatively, if 
clinically stable.  
 
4.7 Withdrawal of MMF/Myfortic 
One month after the fourth infusion (after 1 year posttransplant), participants will be 
evaluated for eligibility for MMF/Myfortic withdrawal.  To be eligible for withdrawal of 
MMF/Myfortic, participants must meet the following criteria:  
• Stable renal function, as defined by Glomerular Filtration Rate (GFR) > 50 mL/min/1.73 
m2. 
• Absence of biopsy-proven or clinically presumed acute rejection episode since 
transplantation. 
• Absence of histologic evidence of acute or chronic rejection, as determined by renal 
biopsy (acute rejection/Banff Grade IA or higher) obtained within two weeks prior to 
commencing withdrawal. 
• Total lymphocyte count > 400/mm3. 
• Completion of all pre-withdrawal tolerance assay studies. 
For participants who meet the eligibility criteria MMF/Myfortic will be withdrawn beginning 
no earlier than 1 year posttransplant.  MMF/Myfortic will be withdrawn in a stepwise fashion 
over a minimum withdrawal period of two months at an approximate rate of 50% of the pre-
withdrawal dose per month.  Clinic visits and frequency are described in the accompanying 
calendar and its footnotes. 
 
4.7.1 Monitoring During and After MMF/Myfortic Withdrawal 
For participants who are withdrawn from MMF/Myfortic, additional laboratory assessments 
(hematology and chemistries) will be obtained according to the following schedule:  
• At the beginning of  withdrawal; then 
• Weekly until MMF/Myfortic is completely withdrawn; then 
• Every other week for the first 6 weeks after complete withdrawal of MMF/Myfortic; then 
• Monthly thereafter. 
A protocol  biopsy will be obtained within 2 weeks prior to commencement of 
MMF/Myfortic withdrawal.  “For-cause” biopsies will also be obtained in the event of a 20% 
increase in serum creatinine that is unexplained and unresolved within 24 hours.  
MMF/Myfortic withdrawal will be halted and/or immunosuppressive medications will be 
reinstituted if any biopsy reveals acute or chronic rejection, or (in the case of protocol 
biopsies not associated with clinical symptoms or laboratory abnormalities) the presence of 
tubulitis.  If a protocol biopsy reveals normal histology or lymphocytic infiltration without 
tubulitis, withdrawal and/or continued absence of immunosuppressive medications will be 
permitted. 
4.8 
Withdrawal of Sirolimus 
At 18 months post-transplant, a second postoperative transplant biopsy will be performed 
(research) and participants will be evaluated for eligibility for Sirolimus withdrawal.  To be 
eligible for withdrawal of Sirolimus, participants must meet the following criteria:  
• Stable renal function, as defined by Glomerular Filtration Rate (GFR) ≥ 50 mL/min/1.73 
m2 (24-hour urine collection for creatinine clearance corrected for body surface area, 
microalbumin, and protein). 

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• Absence of biopsy-proven or clinically presumed acute rejection episode since 
transplantation. 
• Absence of histologic evidence of acute or chronic rejection, as determined by renal 
biopsy (acute rejection/Banff Grade IA or higher) obtained within 2 weeks prior to 
commencing withdrawal. 
• Total lymphocyte count ≥ 400/mm3. 
• Completion of all pre-withdrawal tolerance assay studies. 
For participants who meet the eligibility criteria, Sirolimus will be withdrawn beginning no 
earlier than 18 months post-transplant.  Sirolimus will be withdrawn in a stepwise fashion 
over a minimum withdrawal period of 6 months at an approximate rate of 15-20 % of the 
pre-withdrawal dose per month.  Clinic visits and frequency are described in the 
accompanying calendar and footnotes.   
 
4.8.1 Monitoring During and After Sirolimus Withdrawal 
For participants who are withdrawn from Sirolimus, additional laboratory assessments 
(hematology and chemistries) will be obtained according to the following schedule:  
• At the beginning of Sirolimus withdrawal; then 
• Every other week until Sirolimus is completely withdrawn; then 
• Every other week for the first 4 weeks after complete withdrawal of Sirolimus; then 
• Monthly for 3 years post-operatively and every 2 months thereafter for at least 10 years. 
Biopsies will be obtained within 2 weeks prior to commencement of Sirolimus withdrawal 
and at 12 months after completing withdrawal from Sirolimus.  “For-cause” biopsies  will 
also be obtained in the event of a 20% increase in serum creatinine that is unexplained and 
unresolved within 24 hours.  Sirolimus withdrawal will be halted and/or immunosuppressive 
medications will be reinstituted if any biopsy reveals acute or chronic rejection, or (in the 
case of biopsies not associated with clinical symptoms or laboratory abnormalities) the 
presence of tubulitis.  If a biopsy reveals normal histology or lymphocytic infiltration without 
tubulitis, withdrawal and/or continued absence of immunosuppressive medications will be 
permitted. 
Note:  It will be the practice to follow renal transplant recipients with monthly or bimonthly 
immunologic blood work up to 150 mL and yearly clinic visits (or more frequent if 
necessary) for the life of the transplant. 
 
4.9 
Monitoring of Donors and Healthy Controls 
4.9.1 Short and Long-Term Monitoring of Donors 
Donors will intermittently provide peripheral blood (up to 60 mL [12 teaspoons]) first with 
postoperative clinic visits in the first two weeks after surgery and then for renal chemistries 
and immune assays as outpatients postoperatively at 6 months and yearly intervals for up to 
10 years. Donors will also be asked to provide 25 ml (5 teaspoons) of iliac crest bone marrow 
at 1 or 2 year postoperatively.  
 
4.9.2 Healthy Controls 
4.9.2.1 Inclusion Criteria 
1. Age to 18 – 65 years old.  
2. Male or female of all races and ethnic groups in good health. 
 

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4.9.2.2 Exclusion Criteria 
1. History of systemic disease, e.g. autoimmune disease, diabetes, hypertension, 
infection such as HIV or any disease that requires continuous therapy with prescribed 
medications. 
2. Anyone on current continuous medication including anticoagulants. 
3. Pregnancy. 
 
Up to 110 laboratory volunteer controls will be tested at baseline and followed every 6 
months using peripheral blood monitoring assays in comparison with the HLA identical 
sibling donor/recipient renal transplant pairs. 
HLA identical kidney transplant recipient (see 10.1-10.5). 
Iliac crest bone marrow aspirates (approx. 20-30 mL [4-6 teaspoons] per iliac crest) will 
similarly be tested with a frequency of one bone marrow aspiration (on one or both iliac 
crests) per normal control volunteer. The procedure will not be done more often than once on 
each iliac crest.  
 
4.10 
Recipient and Donor Timelines 
4.10.1  Stem Cell Recipient 
Day 0
Iliac Crest Marrow 
Aspiration, Storage for 
Possible Rescue, then 
Campath-1H
+
Kidney Transplant 
+
Kidney Biopsy
Day 5
Day 4
Month
3
Month 
12
Month
18
Donor Stem 
Cell 
Infusion # 1 
(frozen)
Begin sirolimus 
and withdraw 
tacrolimus 
(overlap sirolimus 

2 weeks)
Leukapheresis
(storage for future 
testing)
Campath-
1H
Donor Stem 
Cell Infusion 
# 2 (fresh)1
Kidney Biopsy, 
then begin 
MMF/Myfortic 
withdrawal (over 
2-4 months)
Kidney Biopsy, 
then begin 
Sirolimus
withdrawal 
(over 6 
months)2
1Two weeks after sirolimus begins.
2If withdrawal criteria outlined in protocol are met.
RECIPIENT
RECIPIENT
Day -30 
Month
1
Month
6
Month
9
Donor Stem 
Cell Infusion 
# 4 (frozen)1
Donor Stem 
Cell Infusion 
# 3 (frozen)
 
Months 2-3
Day 0
Nephrectomy
G-CSF given for 5 days 
prep. prior to 
leukapheresis#1 and #2
Iliac Crest 
marrow 
aspiration
Leukapheresisusing 
peripheral access
DONOR
DONOR
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Renal Chemistries 
Immune Assays 
Mo. 6, Year 1 & 
Yearly thereafter  
Possible Bone 
Marrow Aspiration  
(~25 ml – 5   
teaspoons at 1 or 2 
years postoperatively   
postoperatively) 
Years 2,3, & 5  
Bone Marrow 
Aspiration  (~25 ml); 
kidney biopsy  

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4.11 Study Endpoints 
4.11.1 Primary Endpoint 
The primary endpoint is patient and graft survival measured at the one-year time point post-
transplant. 
 
4.11.2 Secondary Endpoints 
Secondary safety and efficacy endpoints are as follows: 
• Patient and graft survival measured at the three-year time point post-transplant, and 
yearly thereafter; 
• Patient and graft survival measured at the ten-year time point post-transplant; 
• Incidence rate of biopsy-proven acute rejection, defined as a renal biopsy demonstrating 
acute cellular or humoral rejection of Banff Grade IA or greater; 
• Incidence of chronic allograft nephropathy, determined using renal biopsies and labora-
tory values, including 24 urine protein excretion; 
• Incidence of GVHD; 
• Incidence of adverse events associated with renal transplantation and 
immunosuppression, including infections, malignancies (including PTLD), 
thromboembolic events, hyperlipidemia, leukopenia, thrombocytopenia, GI toxicity, and 
cytokine release. 
•  
4.11.3 Definition of Acute Rejection 
For the purposes of this study, an episode of acute rejection will be considered to have 
occurred in the event of: 
• Biopsy-proven rejection: a renal biopsy demonstrates acute cellular or humoral rejection 
of Banff Grade IA or greater; or  
Presumed rejection: in the absence of biopsy-proven rejection, the participant is treated with 
augmentation of immunosuppression for an unexplained 20% increase in serum creatinine. 
 
4.12 Study Population  
Adult recipients of living-related (HLA identical) donor recipient pairs will be eligible to 
participate in this study.  Because safety has not yet been established, participants under the 
age of 18 years will not be included in the study.  Participants who meet the eligibility 
criteria may be enrolled with no restrictions due to gender or race. 
4.13 Screening and Enrollment 
This research study will be explained in lay language to each potential research participant.  
The participant will sign an informed consent prior to any screening study procedures.  All 
activities (please see study Schedule of Events) performed in conjunction with this study will 
be performed in an unblinded manner. 
 
4.14 Criteria for Discontinuation of Protocol Regimen 
Patients who experience an acute rejection episode or an adverse event related to Campath-
1H, tacrolimus, sirolimus, MMF, or a combination of these medications, may, at the 

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discretion of the Investigator, receive alternate FDA approved immunosuppressive 
medications and/or dosing regimens appropriate for the participant’s clinical condition.   
 
4.15 
Criteria for Suspending Enrollment into the Study and/or Modification of 
Withdrawal of Immunosuppressive Medications 
The medical monitor and/or statistician will review selected safety and efficacy criteria on 
an ongoing basis. Study enrollment and withdrawal of immunosuppressive medications will 
be suspended pending expedited review of all pertinent data if any one of the following 
occurs: 
• Any participant experiences biopsy-proven acute rejection at any time during the study. 
• Any participant experiences chronic allograft nephropathy at any time during the study. 
• Any participant experiences GVHD at any time during the study. 
• Any participant experiences graft loss during the first 12 months of the study. 
• Any participant dies during the first 12 months of study, where the death is attributed in 
any way to treatment received in this study. 
 
In the event of the occurrence of any of the above, pertinent study data will be expeditiously 
reviewed by the NU Institutional Review Board, and once the protocol is approved by the 
following groups: the NU/NMH CRU Data Safety Monitoring Board (DSMB) an 
independent extra institutional DSMB, the FDA, and the NIDDK. After this review, the 
Investigator and these agencies will jointly make a final determination regarding 
continuation, modification, or termination of the study. 
 
5 
Selection and Withdrawal of Participants 
5.1 
Inclusion Criteria 
1. Patient has been fully informed, has signed a dated IRB approval informed consent form 
obtained directly by the P.I., Co-P.I., sub-investigator and/or research nurse and is willing 
to follow study procedures for the extent of the study (3 years). 
2. Recipient must have a hematocrit of 33%, and a hemoglobin of 11.0 g/dL. 
3. Age 18-65 years.  
4. Weight > 40 kg. 
5. Primary renal allograft: living-related (HLA identical donor).  
6. Negative B-cell and T-cell cytotoxic and flow cytometry cross-match, and panel-reactive 
antibodies of < 10%. 
7. Women of childbearing potential will be required to have a negative qualitative serum 
pregnancy test.  See also #11, below. 
8. Patients are to be studied equivalently as they become available for transplantation using 
these criteria, without regard for gender, race, or ethnicity. 
9. Normal echocardiogram with an ejection fraction >50%.  
10. Male participants with reproductive potential must agree to use approved methods of 
birth control during treatment with Campath-1H and for a minimum of 6 months 
following their last dose of Campath-1H. Female participants of childbearing potential 
must agree to use approved methods of birth control for the duration of their participation 
in the study. 
11. Patient must agree to be followed every 2 months after year 3, for up to 10 years. 

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14.  Up to 25 laboratory volunteers will be tested at baseline and followed every 6 months 
using peripheral blood and bone marrow monitoring assays with their HLA identical 
siblings. Up to 25 other volunteers (without HLA identical siblings) will also be tested 
once as controls for blood and bone marrow assays.  These assays will be similar to those 
performed on the stem cell infused HLA identical kidney transplant recipient (see 10.1-
10.5). 
 
5.2 
Exclusion Criteria 
1. Patient has previously received or is receiving an organ transplant other than a kidney.  
2. Patient is receiving an ABO incompatible donor kidney.  
3. Recipient or donor is ELISA positive for human immunodeficiency virus (HIV), antibody 
positive for hepatitis C, or surface antigen positive for hepatitis B. 
4. Recipient or donor is positive for TB (or under treatment for suspected TB), or has had 
previous exposure to TB (positive Mantoux), who has not undergone and acceptable 
course of treatment. 
5. Patient has a current malignancy or a history of malignancy (within the past 5 years), 
except non-metastatic basal or squamous cell carcinoma of the skin, or carcinoma in situ 
of the cervix that has been treated successfully.  
6. Patient has uncontrolled concomitant infections and/or severe diarrhea, vomiting, active 
upper gastro-intestinal tract malabsorption or an active peptic ulcer or any other unstable 
medical condition that could interfere with study objectives.  
7. Patient is currently receiving an investigational drug within 30 days prior to transplant.  
8. Patient is currently receiving any immunosuppressive agent.  
9. In the judgment of the investigator, it is anticipated that the patient will be unable to take 
medications orally or via nasogastric tube by the morning of the second day following 
completion of the transplant procedure (i.e., skin closure).  
10. Concurrent use of warfarin, fluvastatin, astemizole, pimozide, cisapride, terfenadine, or 
ketoconazole.  
11. Patient has a known hypersensitivity to tacrolimus, Campath-1H, Thymoglobulin, 
daclizumab (Zenapax®), sirolimus, MMF or corticosteroids.  
12. Patient is pregnant or lactating.  
13. Patients with a screening/baseline total white blood cell count <4000/mm3; platelet count 
<100,000/mm3.  
14. Patient is deemed unlikely to comply with the visits scheduled in the protocol. 
15. Patient has any form of substance abuse, psychiatric disorder or a condition that, in 
opinion of the investigator, may invalidate communication with the investigator.  
16. It is expected that tacrolimus cannot be instituted for longer than 5 days postoperatively.  
17. Patients with a PRA value >10% at any time prior to enrollment. 
18. Patients with Graves disease will be excluded unless previously adequately treated with 
radioiodine ablative therapy. 
19. EBV negative recipients of a EBV positive donor. 
20. Patients with a history of idiopathic thrombocytopenic purpura (ITP) or thrombotic 
thrombocytopenic purpura (TTP). 
 

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5.3 
Participant Withdrawal Criteria 
5.3.1 Premature Termination from the Study 
Participants may be prematurely terminated from the study; however, documentation and 
follow-up for safety will continue for one year even if an individual participant is terminated 
from the study.  Premature termination of an individual participant will take place should any 
of the following occur: 
1. Informed consent is withdrawn. 
2. Adverse events of grade 3 or higher are documented (according to full NIH guidelines in 
minimizing the risk and in Equipoise considerations). 
3. In case of pregnancy. 
4. In case of medical non-compliance. 
5. In case of other serious intervening illnesses such as infection, GVHD, or posttransplant 
lymphoma. 
6. In case of rejection or other adverse events occur requiring changing to our standard 
immunosuppressive protocols.   
 
5.3.2 Replacement of Participants who Discontinue Study Treatment or who 
Prematurely Terminate from the Study 
Participants who discontinue study treatment or who prematurely terminate from the study 
will not be replaced. 
 
6 
Study Medication 
6.1 
Campath-1H: Formulation, Packaging, and Labeling 
Although not considered an experimental drug, for the purposes of the study Campath-1H 
will be supplied by Genzyme Corporation, San Antonio, TX. Campath-1H is supplied as a 
purified preparation diluted in phosphate buffered saline (PBS) with 0.05 mmol 
Ethylenedinitrilo Tetraacetic Acid (EDTA).  Between 80 is added to a concentration of 
0.01%.  The final product is a clear, colorless isotonic solution free of visible particulate 
matter.  The ampules will contain 10 mg of antibody in 1mL of sterile PBS at a concentration 
of 10 mg/mL.  Campath-1H should be stored, protected from light, in a refrigerator between 
2 and 8C.  
 
Intravenous Campath-1H will be diluted in 100 cc of 0.9% normal saline or 5% Dextrose and 
water (D5W) and administered intravenously over 2 hours.  Campath-1H must be filtered 
with a sterile, low-protein binding, 5-micron filter prior to dilution. 
 
6.2 
Prophylactic Medications 
6.2.1 Pneumocystis carinii (PCP) 
Trimethoprim / sulfamethoxazole (Septra/Bactrim) one single-strength tablet will be 
administered daily  for the first 6 weeks, then 3 times a week for the duration of the study, as 
tolerated.  If unable to tolerate or if allergic, dapsone will be substituted after a Glucose –6- 
Phosphate Dehydrogenase hemolysis (G-6-PD) blood test has been shown to be normal. 
  
6.2.2 Cytomegalovirus (CMV)  
Valganciclovir (Valcyte) 450mg will be administered daily as tolerated for the first three 
months posttransplant.  The valgancyclovir dosing will be based on the participant’s 
calculated creatinine clearance.  Subsequent dose reductions may be made at the investigator 

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discretion.  Valtrex will be given, as tolerated, if patient does not tolerate Valcyte or 
becomes leukopenic. 
 
6.2.3 Fungal 
Nystatin or Clotrimozole will be administered for fungal prophylaxis for the first 3 months 
posttransplant. 
 
7 
Study Procedures 
A detailed schedule of events (Up to year 3) for this study is provided in Appendix 1.  
 
Year 4-10 follow-up:  will be conducted by annual EMR review of Standard of Care (SOC) 
laboratory and clinical follow up on the subject's transplant anniversary date. The study staff 
will also contact the subjects once annually (around patients’ anniversary date) to review 
adverse events. The annual subjects findings will be communicated and signed off by the PI 
regularly.  
 
7.1 
Protocol and For-cause Biopsies 
Protocol biopsies will be obtained 2 weeks prior to commencement of MMF withdrawal, 2 
weeks prior to Sirolimus withdrawal, and 1, 2, and 5 years after Sirolimus withdrawal.  “For-
cause” biopsies will also be obtained in the event of a 20% increase in serum creatinine that 
is unexplained and unresolvefd within 24 hours.  Withdrawal will be halted and/or 
immunosuppressive medications will be reinstituted if any biopsy reveals acute or chronic 
rejection, or (in the case of protocol biopsies not associated with clinical symptoms or 
laboratory abnormalities) the presence of tubulitis.  If a protocol biopsy reveals normal 
histology or lymphocytic infiltration without tubulitis, withdrawal and/or continued absence 
of immunosuppressive medications will be permitted. 
 
7.1.1     Protocol Bone Marrow Aspirations 
Protocol bone marrow aspirations will be obtained at 5 years after Sirolimus withdrawal. 
 
7.2 
Treatment of Acute Rejection 
Mild (Banff IA):  3 daily I.V. pulses of 500 mg of methylprednisolone, followed by an oral 
taper over approximately 2-3 weeks.  Moderate (Banff IB) or severe (Banff IIA or greater):  
Thymoglobulin® (1 mg/kg per day for 5-10 days).  Treatment for acute rejection may be 
modified by the Investigator or as clinically indicated. 
 
7.3 Guidelines for Treating Neutropenia 
If a patient’s absolute neutrophil count (ANC) is <700 cells/µL, the following guidelines are 
recommended: 
• 
Reduce the prophylactic dose of valganciclovir. 
• 
Stop administration of Septra SS and replace with inhaled pentamidine. 
If the participant’s ANC is <500 cells/µL, the following guidelines are recommended: 
• 
Consider discontinuation of valganciclovir. 
• 
Obtain a CMV PCR. 
• 
If fever is associated with neutropenia obtain a hematology and infectious disease 
treatment consultation and initiate appropriate antibiotics and antifungal agents as per 
consultation. 

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• 
Consider fluoroquinolones. 
• 
Consider GCSF (neupogen). 
• 
If currently taking sirolimus, obtain a new whole-blood sirolimus trough level (and also 
refer to the most recent available sirolimus trough level to avoid delay in patient 
management), and if the sirolimus levels are >12 ng/mL, then consider holding 1 to 2 
doses (based on clinical judgment).  If the sirolimus levels are <12 ng/mL, then use 
clinical discretion in continuing on sirolimus. 
• 
Consider restarting sirolimus as soon as ANC count is >500 cells/µL. 
 
The participant’s safety is of utmost importance.  Local clinical treatment decisions take 
precedence over recommended guidelines. 
 
7.4 
Diagnosis and Treatment of Graft Versus Host Disease 
In the current Campath-1H induction protocol, by the very use of the antibody and its half-
life of 12 days with effects felt up to one year, it is conceptually highly unlikely that in the 
recipient non-myeloablated environment that contaminating donor post-thymic T cells can do 
this.  Furthermore, it is perhaps more likely that a mixed (mutually tolerant) chimeric state 
could ensue if macrochimerism of >10% donor immunohematologic cells would occur.  
GVHD would be especially unlikely under recipient continuous immunosuppressive therapy, 
essentially for 2 years after the second stem cell infusion.  Additionally, by purifying CD34+ 
cells post-thymic, T cells in the inoculum are minimized. 
 
Nonetheless, as a safeguard, it is planned to store one unit of iliac crest recipient bone 
marrow taken at transplant surgery before Campath-1H is started, so that it can be re-infused 
any time during the long observation period in order to reverse GVHD if needed.  In 
addition, our collaborator Dr. Richard Burt, Director of Immunotherapy at Northwestern, will 
be following these patients and has diagnostic and therapeutic protocols in place that 
incorporate the following. 
 
7.4.1 Diagnosis of Acute GVHD 
Acute GVHD generally develops within the first three months after transplantation and 
appears as a characteristic dermatitis often accompanied by hepatic cholestasis and enteritis.  
The clinical appearance of GVHD may be mimicked by drug reactions.  Therefore, 
documentation by skin biopsy will be performed.  Liver biopsy can be helpful but often 
cannot be done because of clinical contraindications such as thrombocytopenia. 
 
7.4.2 Treatment of GVHD 
Grade I – II GVHD will be treated with Solumedrol (2 mg/kg) and Prograf.  In cases of 
moderate to severe GVHD, Zenapax and anti-TNF antibody (Remicade) will be utilized. 
 
8 
Assessments of Efficacy and Safety 
8.1 
Safety and Efficacy Parameters 
In the absence of medical contraindications, all acute rejection episodes will be confirmed by 
biopsy.  Biopsies will be histologically evaluated at the Northwestern Memorial Hospital, 
utilizing the Banff criteria. 
 

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For the purposes of this study, an episode of acute rejection will be considered to have 
occurred in the event of: 
Biopsy-proven rejection: a renal biopsy demonstrates acute cellular or humoral rejection of 
Banff Grade IA or greater; or  
Presumed rejection: in the absence of biopsy-proven rejection, the participant is treated with 
augmentation of immunosuppression for an unexplained 20% increase in serum creatinine.     
Graft loss is defined as the institution of chronic dialysis (at least 6 consecutive weeks, 
excluding participants with delayed graft function), transplant nephrectomy, or 
retransplantation. 
 
For this study, severe acute rejection is defined as that which is histologically evaluated as 
Type IIA or greater utilizing the Banff criteria. 
 
Renal function will be evaluated through the measurement of serum creatinine monthly 
posttransplant. 
 
Adverse events to include infections and malignancies and participant information to include 
participant weight and resting blood pressure will be collected on study case report forms 
throughout the study. For purposes of this protocol, rejection, graft loss, and status of graft 
function will not be recorded on the adverse event case report form. These events will be 
collected on specialized case report forms. 
 
9 
Adverse Event Collection and Reporting 
Adverse events that are classified as serious, according to the definition of regulatory 
authorities, must be rapidly and adequately reported to the regulatory authorities mentioned 
above.  This section provides definition of the types of adverse events and outlines a process 
for the appropriate reporting and follow-up procedures.  Information in this section was 
obtained from the ICH guideline for Good Clinical Practice (mentioned previously), ICH 
Guideline E2A: Clinical Safety Data Management: Definitions and Standards for Expedited 
Reporting, and the Common Toxicity Criteria Manual Version 3.0 (August 9, 2006). 
 
9.1 
Definitions 
9.1.1 Adverse Event 
An adverse event is any occurrence or worsening of an undesirable or unintended sign, 
symptom (including an abnormal laboratory finding), or disease temporally associated with 
the use of a medicinal product, whether or not considered related to the medicinal product. 
Throughout the study-defined collection period, the Investigator must record all adverse 
events on the appropriate adverse event form, regardless of the severity or relationship to 
study medication or procedure. The Investigator should treat participants with adverse events 
appropriately and observe them at suitable intervals until the events resolve or stabilize.  
Adverse events may be discovered through:  
• 
Observation of the participant;  
• 
Questioning the participant; 
• 
Unsolicited complaint by the participant; or 
• 
Discovery of abnormal clinical laboratory values or abnormal results of other evaluations 
(radiographs, ultrasound, ECG, etc.). 
In questioning the participant the questioning should be conducted in an objective manner.   

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In the event of an abnormal laboratory value, the test should be repeated until it returns to 
normal or can be explained and the participant’s safety is not at risk.  Clinically significant 
laboratory abnormalities as determined by the Investigator must be recorded as adverse 
events, as well as recorded on the appropriate laboratory evaluation form(s). 
As indicated in the Appendix I, AEs will be collected from the start of the study through 
Month 36. AEs that are unresolved at the time that the participant completes this period of 
the study will be followed until they resolve or for a maximum of 30 days. 
 
9.1.2 Serious Adverse Event 
A serious adverse event (SAE) or reaction is defined as any adverse event occurring at any 
dose that suggests a significant hazard, contraindication, side effect or precaution. This 
includes, but may not be limited to any of the following events:  
• Death: A death occurring during the study or which comes to the attention of the 
Investigator during the protocol-defined follow-up after the completion of therapy, 
whether or not considered treatment-related, must be reported; 
• Life-threatening: Any adverse therapy experience that places the patient or participant, in 
the view of the Investigator, at immediate risk of death from the reaction as it occurred; 
• Inpatient hospitalization or prolongation of existing hospitalization; 
• Persistent or significant disability/incapacity; 
• Congenital anomaly/birth defect; 
• Other conditions as specified in the protocol; 
• An event that required intervention to prevent permanent impairment or damage. An 
important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical 
judgment, it may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
 
SAEs will be collected from the time informed consent is obtained, to 30 days after study 
completion or for a minimum of one year after a participant prematurely withdraws from the 
study or for the duration of the funding period. 
 
However, patients will be followed for collection of events such as death, graft survival, 
resumption of immunosuppressive medications, the development of life-threatening 
infections, and the development of cancer during a 10-year long-term follow-up. 
Regardless of the relationship of the adverse event to study drug, the event must be reported 
as a serious adverse event if it meets any of the above criteria. 
 
9.1.3 Unexpected Adverse Event 
An adverse event is considered unexpected when the nature (specificity) or severity of the 
event is not consistent with applicable product information, such as safety information 
provided in the package insert, investigational plan, investigator’s brochure, protocol or 
informed consent document. 
 

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9.2 
Grading of Adverse Events 
9.2.1 Toxicity Grading of Adverse Events 
Toxicity grades are assigned by the study site to indicate the severity of adverse events 
occurring in study participants. These have been adopted from the National Cancer Institute 
(NCI) Common Toxicity Criteria (CTC) for application in adverse event reporting.  The 
purpose of using the NCI-CTC system is to provide a standard language to describe 
toxicities, to facilitate tabulation and analysis of the data, and to facilitate the assessment of 
the clinical significance of all adverse events. The NCI-CTC provides the following grades 
and descriptions in the NCI-CTC Manual (Version 3.0, dated 8/9/06).  Adverse events should 
be recorded and graded 1 to 5 according to the NCI-CTC grades provided below: 
Grade 1 = Mild adverse event. 
Grade 2 = Moderate adverse event. 
Grade 3 = Severe and undesirable adverse event.  
Grade 4 = Life-threatening or disabling adverse event. 
Grade 5 = Death. 
Note: In contrast to the CTC guidelines provided in the NCI-CTC Manual (Version 3.0) all 
adverse events are to be reported and graded, whether or not related to disease progression or 
treatment.   
 
9.2.2 Relationship to Study Drug 
The relationship or attribution between an adverse event and an investigational product is 
determined by the site Investigator or sub-Investigator and recorded on the appropriate Case 
Report Form and/or SAE Reporting Form. The NCI-CTC provides the following descriptors 
and definitions (one category classified as unrelated [Code 1] and 4 categories classified as 
related [Codes 2-5]) for assigning an attribution to each adverse event as described below: 
ATTRIBUTION OF ADVERSE EVENTS 
Code 
Descriptor Definition 
“Unrelated” Category Code (1 Category): 
1 
Unrelated 
The adverse event is clearly not related to the investigational 
agent(s) 
“Related” Category Codes (4 Categories): 
2 
Unlikely 
The adverse event is doubtfully related to the investigational 
agent(s) 
3 
Possible 
The adverse event may be related to the investigational agent(s) 
4 
Probable 
The adverse event is likely related to the investigational agent(s) 
5 
Definite 
The adverse event is clearly related to the investigational agent(s) 
 
The Investigator’s determination of drug-relatedness (attribution) for each adverse event 
should be recorded in the source documentation. 
For additional information, consult the Common Toxicity Criteria Manual and the Common 
Toxicity Criteria Document at the following URL: http://ctep.cancer.gov/reporting/ctc.html.  
 

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9.3 
Serious Adverse Event Reporting 
The following process for reporting a serious adverse event will ensure appropriate reporting 
compliance with the ICH guidelines. 
 
9.3.1 Serious Adverse Event Identification and Determination of Reporting Timeline 
When an Investigator identifies a serious adverse event (as defined above), the Investigator 
must notify the NU IRB and/or other Reporting Centers (the NUCRU, FDA, DSMB, and 
NIDDK) of the serious adverse event within 24 hours of discovery.  In addition, these events 
must be entered on the Serious Adverse Event Form and the Adverse Event CRF.  
Three possible reporting scenarios (to the appropriate health authorities) could arise after 
assessment of the event: 
No requirement to report.  This would occur if the adverse event is deemed not serious by 
protocol definition. 
 
Standard reporting is required (report in IND annual report).  This would occur if the adverse 
event were classified as one of the following: (a) serious, expected and drug related; (b) 
serious, expected and not drug related; or (c) serious, unexpected and not drug related. 
Expedited reporting is required.  This would occur if the adverse event is considered serious, 
unexpected and drug related.  These events must be reported by the Sponsor to the 
appropriate health authorities within 15 days unless the event is fatal or life- threatening, the 
latter must be reported within 7 days.  
 
The PI in any event, must report the serious adverse event to the NU IRB as mandated by 
the NU IRB. 
 
9.3.2 Reporting Serious Adverse Events to the Data and Safety Monitoring Board 
A Transplant Data and Safety Monitoring Board (DSMB) will be provided listings of all 
SAEs on an ongoing basis. Further, the DSMB will be informed of expedited SAEs at the 
same time as regulatory authorities. 
 
9.4 
Pregnancy (SAE Reporting Requirements) 
Any pregnancy that occurs during a clinical study with an investigational drug must be 
reported as an SAE for tracking purposes only. All pregnancies that are identified during this 
study need to be followed to conclusion and the outcome reported. Female participants 
should immediately inform the Investigator of any pregnancies and should be instructed by 
the Investigator to stop taking study medication.  The Investigator should report all 
pregnancies within 24 hours (as described above in SAE Reporting) using the SAE form. The 
Investigator should counsel the participants and discuss the risks of continuing with the 
pregnancy and the possible effects on the fetus. Monitoring of the participants should 
continue until the conclusion of the pregnancy, and a follow-up SAE Reporting Form should 
be submitted detailing the outcome.  
 
9.5 
Updating Source Documentation 
Documents describing the safety profile of a drug, such as the investigator’s brochure, should 
be amended, as needed, to ensure that the description of safety information adequately 
reflects any new clinical findings. Until these documents are updated, expedited reporting is 
required for additional occurrences of the reaction. 

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10 
Tolerance Assay Studies 
A series of assays of chimerism analyses and tolerance mechanism are to be performed. 
10.1 
Cytokyne Gene Polymorphism (CGP)-PCR-Flow Measurements of DHSC 
Chimerism 
Hypothesis: We propose that macrochimerism brought about by DBMC stem cell infusions, 
together with Campath-1H treatment for making “space” in recipient central and peripheral 
immune compartments, will be a reflection of the development of donor-specific 
unresponsiveness. Thus, after treatment with Campath-1H, these assays (and other 
measurements of DBMC chimerism) should demonstrate significantly higher levels of 
chimerism (macrochimerism) than in our previous DBMC protocols1,2,41, especially since 
CD34+ cells are not sensitive to Campath-1H lysis. 
 
Plan: The CGP-PCR-Flow assays42,43 (using cytokine polymorphism) as well as VNTR 
assays of PBMC subsets will be used to sequentially measure donor immunohematopoietic 
cell chimerism in peripheral blood and iliac crest marrow, i.e., of donor and recipient CD3+, 
CD4+, CD8+, CD34+, CD19+, CD16/56+, and CD14+ cells, according to the following 
time-table: 
1. Intraoperatively (baseline control values), before Campath-1H administration, but after 
anesthesia induction in: (a) donor peripheral blood and donor iliac crest marrow, and (b)  
recipient peripheral blood and recipient iliac crest marrow. 
2. In (a) recipient peripheral blood on post-operative days 4, 6 (1 day post-DBMC infusion), 
21 and 28, then monthly for months 2-12 and every six months thereafter, and (b) 
recipient iliac crest marrow at 6 months, 1-year and yearly thereafter. 
3.  
It will be of interest to determine with the CGP-PCR flow assay whether or not the relatively 
more rapid return of certain recipient immunohematopoietic subsets, i.e., relatively Campath-
1H resistant CD14+ and CD16/56+ cells, influences the presence of specific donor chimeric 
subsets, i.e., CD34+ cells or CD3+ cells, as they regenerate (compared with DBMC-infused 
recipients tested in previous studies). 
 
Alternatively: If there is no difference using CGP-PCR-Flow analysis between the DHSC-
infused Campath-1H group and patients in previous studies, there may well be a greater 
difference in the ratio of CD34+ to CD3+ donor chimeric cells in the stem cell-infused 
group.1,2 This in itself will be of interest for possible future protocol design and other 
mechanistic ex vivo assays. 
 
Alternatively: If significant macrochimerism (>10%, vide supra) does not develop under 
Campath-1H treatment, the assays and clinical studies described in the succeeding sections 
may well show differences between this stem cell-infused group and those previously 
reported, so at to test whether immunosuppressive withdrawal is still be possible. 
 
10.2 Sequential Measurement of Regulatory Cells and their Products 
Ex vivo immune assessment:  Such cells in the peripheral blood and (bone marrow) at 6, (12), 
18, (24), 30, (36), 42, (48), 54, and (60) months postoperatively. [( ) = iliac crest marrow 
samples] 
 

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Hypothesis:    Regulatory cells of donor and recipient phenotypes that develop as a result of 
chimerism in the DBMC-infused group of renal transplant recipients are causative agents of 
specific hyporesponsiveness to donor alloantigens and can be quantitated sequentially as a 
means of assessing the effect of immunosuppressive withdrawal.  We will use ELISPOT 
assays, flow cytometric analysis, and the micro-CML that requires fewer cellular reactants.  
We will monitor the renal transplant recipients from the DBMC-infused groups for their anti-
donor immune status and T cell regulatory function.  Greater immunoregulatory activity is 
expected in the Campath-1H donor stem cell infusion group than tested in previous DBMC-
infused patients.  Studies by Heeger and colleagues have suggested that IFN-  or Granzyme-
B producing cells, as detected by ELISPOT in allograft recipients, provide an ex vivo 
reflection of the evolving in vivo donor reactive immune response.44 The antigen specific 
production of these cytokines in this short-term assay represents the fingerprint of a pre-
viously primed T cells of the CD45RO+ and CD45RA- memory phenotype, i.e., a putative 
escape from the regulatory or clonal deletion effects of the tolerance protocol.  However, 
production of IL-4, IL-10 and other TH2 cytokines may represent the presence of regulatory 
cells. 
 
10.2.1 ELISPOT 
Plan: Ninety-six-well ELISPOT plates (Cellular Technology Ltd, Cleveland, OH) are coated 
with 100 l per well of capture antibodies for IFN- , Granzyme-B, IL-4 or IL-10 (BD 
Sciences) in PBS overnight at 4ºC.  The plates are then blocked with 150 l of PBS-1% BSA 
(Sigma) per well and washed three times with PBS.  Usually 300,000 recipient responder 
cells per well are stimulated with 300,000 (donor) stimulator cells per well.  
Phytohemagglutinin is added to selected wells as a positive control. Final volume for all 
assays is 200 l per well.  Control wells contain responder cells or stimulators plus medium 
alone.  After incubation at 37ºC for 24 hr, the plates are washed three times with PBS, then 
four times with PBS-Tween (0.025%).  Next, 100 l of biotinylated detection antibodies for 
IFN- , Granzyme-B, IL-4 or IL-10 (BD Sciences) in PBS-Tween-1% BSA are added to the 
wells overnight at 4ºC.  On the following day, the plates are washed three times with PBS-
Tween. Streptavidin-HRP (PharMingen) is plated at optimal dilution in PBS-Tween-BSA for 
90 min at room temperature. The spots are developed using 800 l of 3-amino-9-
ethylcarbazole (Pierce; 10 mg/mL in N, N-dimethylformamide) freshly diluted into 24 mL 
0.1M sodium acetate (pH 5.0), filtered through a 0.45-mm filter and mixed with 12 l of 
H2O2 (200 l/well).  After the plates are dried, the resulting spots are counted on a computer-
assisted ImmunoSpot image analyzer in use in our laboratories (Cellular Technology 
Ltd),that has 100% reproducibility when repeatedly counting the same well using single, 
defined criteria.  Results are presented as mean values of ELISPOT detected in triplicate 
wells containing responder PBL plus mitogen or donor stimulator cells, after subtracting the 
response of wells with responder cells or donor cells alone (<15 spots per 300,000 cells in 
each case). 
 
10.2.2 Micro-CML Assays 
These will be performed using recipient responding PBL against both donor and 2DR 
mismatched indifferent (third party) cells of a selected laboratory volunteer.30 
Inhibition in Micro-CML or in MLR by RdD cells will be indicative of regulatory activity by 
the donor chimeric cells present in the recipient and, by RdR cells, will be suggestive of the 
presence of “infectious tolerance”. The presence of this regulatory activity will be confirmed 

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with the use of cryopreserved recipient pre-transplant PBL as responders and posttransplant 
RdD and RdR cells as well as pre-transplant recipient cells as modulators. Inhibition under 
these conditions will indicate that chimerism can drive the recipient to develop regulatory 
cells that act on autologous anti-donor immune responses. 
 
Alternatively: In recipients who show micro-CML and MLC unresponsiveness to the donor, 
the presence of possible split or linked tolerance will be evaluated.45 The recipient PBL will 
be stimulated with irradiated PBL from laboratory volunteers who have common donor HLA 
antigens and at least 1 indifferent HLA antigen (from both donor and recipient), compared 
with volunteers with totally different HLA genes, in the presence of graded numbers of 
purified RdD and RdR cells. Inhibition of these semi-allogeneic vs. fully allogeneic 
responses will be indicative of linked suppression. 
 
For each of these assays, 40 ml of recipient blood and 20 ml of bone marrow are needed at 
each testing interval.  They will be performed at the time of the PCR-Flow assays when bone 
marrow is obtained (5 ml of marrow). 
 
10.2.3 Treg and Immunophenotyping Assays 
We are now using the Treg MLR functionally in invitro assays testing peripheral blood from 
our recipients as well as immunophenotyping for Tregs. 
 
10.3 Donor Reactive Cells or Regulatory Cells in the Kidney Allograft 
Hypothesis:    Either donor reactive or regulatory cells may be sequestered in the allograft 
itself. Regulatory reactivity can be assessed with more frequency in the peripheral circulation 
(or bone marrow), using the approaches described above, and (less frequently) in the 
allograft by monitoring infiltrating cells in biopsies. With the sequential (yearly) renal trans-
plant biopsies assessing the effect of immunosuppressive withdrawal, it will be possible to 
assay this graft-associated lymphoid compartment for such reactivity. 
 
Plan:  Protocol renal transplant biopsies will be taken at yearly intervals, i.e., before, during 
and after planned immunosuppressive therapy withdrawal.  In addition to histopathological 
examination, a portion of the biopsies will be placed in culture in medium containing 50 
U/ml rIL-2 and 50% MLR supernatant. 46 During rejection, lymphocytes usually grow out of 
the biopsy within 48 hours.46  We will keep the biopsy cultures for at least 1 week, and 
characterize them for donor vs. recipient HLA genomic DNA by PCR (or even by cellular 
PCR-Flow) at 48 hr, 72 hr, or 7 days after the initiation of the biopsy culture.  If sufficient 
numbers of cells are available, their ability to kill donor cells or regulate killing in a modified 
micro-CML assay and to produce Granzyme-B, IFN- , IL-4 and IL-10 in the ELISPOT 
assay will be monitored. 
 
For PCR amplification and reverse probe hybridization of CGP profiles using the Inno-
genetic LIPA (Line Probe Assay), approximately 100 cells will be needed for the genomic 
DNA extraction and purification. If a mixed genotype is resolved, a quantitation analysis will 
be accomplished by Sequence Specific Priming (SSP) of mismatched donor/recipient alleles.  
The recipient amplified product will be compared to a standard to ascertain the quantity (%) 
of recipient versus donor alleles.  From as few as 1000 of these cells, the profile of messen-
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time PCR quantitation with cytokine primers.47,48  The absence of CTL or Granzyme-B and 
IFN-  producing cells and/or a high ratio of donor to recipient genomic MHC DNA activity 
growing out from the biopsies will be considered indicative of donor specific 
unresponsiveness, and the presence of IL-4 or IL-10 producing cells will be indicative of 
regulatory cells. 
 
Alternatively:   If no cells grow out (and no histopathology is present), this will be an even 
clearer sign of alloimmune non-reactivity. 
 
10.4 
Absence of Recipient Donor-reactive Cytotoxic T cell Precursors and/or the 
Presence of Regulatory Cells in the Peripheral Circulation  
If the recipient demonstrates donor-specific unresponsiveness in the ELISPOT and micro-
CML assays, limiting dilution analysis for the enumeration of CTLp will be performed. A 
precursor frequency of less than 10 donor reactive CTLp in 1x106 responder PBL will be 
taken as unresponsiveness.  Additionally, the shape of the limiting dilution analysis curve 
would also indicate the presence of regulatory cells in the responding PBL. 
 
10.5 
Mixed Lymphocyte (Dendritic) Cell Culture (ML(D)C), Treg MLR, and In-vivo 
DTH Reactions 
These assays are to be performed on the recipient to detect unresponsiveness and regulatory 
cells in iliac crest marrow aspirates and peripheral blood. 
 
10.6    Sensitive Alloantibody Assays 
Since, the presence of antibodies to donor MHC antigens has been associated with rejection 
and graft destruction in organ transplant recipients49 and, the absence of such donor reactive 
antibodies is a prerequisite for allograft acceptance, we propose that monitoring serial serum 
samples even from HLA identical recipients for expanded HLA specificities, and non-HLA 
epitopes and RBC and endothelial cell line using the Flow technique should help delineate 
this alloimmunoreactive predisposition.  This will also be performed in collaboration with 
laboratories of Dr. Paul Terasaki in Los Angeles, California. 
 
11.0  Additional genomic studies on paraffin embedded transplant biopsy samples 
These samples will have been obtained at the time of the transplant biopsies and will be 
processed and sent to our collaborators at the Scripps Institute in La Jolla, California for 
genomic assays on biopsy material already obtained.  
 
The purpose of the additional genetic studies on paraffin embedded transplant biopsy 
samples is to obtain a biomarker that is indicative of kidney tolerance compared to kidney 
rejection in renal transplant patients.  
 
12.0  Statistical Considerations and Analytical Plan 
12.1  Study Objectives 
12.1.1 Primary Objective 
The primary objective of this trial is to investigate the safety and efficacy of a treatment 
regimen consisting of induction therapy with Campath-1H and steroid-free low dose 
maintenance immunosuppression (consisting of methylprednisolone, MMF, tacrolimus, and 
sirolimus), followed by complete withdrawal of immunosuppression beginning at one year, 

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at a minimum, posttransplant, in recipients of living-related (HLA identically  matched) 
donor kidney transplants who have also been given infusions of purified DBMCs. 
 
12.1.2 Secondary Objectives  
To assess the utility of various assays and biopsy information as putative tolerance markers 
in these kidney transplant recipients: 
• To use the short tandem repeat (STR) assay both in recipient peripheral blood on a 
monthly basis and in iliac crest marrow at 6 months and yearly, in order to measure donor 
cell multi-lineage chimerism; 
• To use the newly developed cell-mediated lympholysis (CML) microassay and enzyme-
linked immunospot (ELISPOT) assay to demonstrate sequentially increasing donor and 
recipient chimeric marrow and peripheral blood cell immunohematologic subset 
regulatory effects; 
• To study lymphoid cells derived from yearly kidney biopsies (i.e., before and after 
planned withdrawal of immunosuppression) 
• To follow sensitive alloantibody assays;  
. 
12.2 
Endpoints 
12.2.1 Primary Endpoint 
The primary endpoint is patient and graft survival measured at the one-year timepoint post-
transplant. 
 
12.2.2 Secondary Endpoints 
Secondary endpoints to be evaluated include those parameters outlined and defined in 
Section 4.10.2 Secondary Endpoints. 
 
12.3 
Sample Size 
Ad described above. 
 
12.4 
Randomization and Blinding  - none. 
 
12.5 
Statistical Analysis 
12.5.1 Analysis Samples 
The following analysis samples will be utilized for safety and efficacy analyses: 
Safety sample: All participants who receive any form of study therapy will be included in all 
safety analyses. 
Intent-to-treat (ITT) sample: All participants will comprise the ITT sample. All efficacy 
endpoints will be analysis on an ITT basis. 
Per Protocol (PP) sample: All participants without major protocol violations who are not 
replaced due to technical reasons and who have adequate efficacy assessment data will 
comprise the PP sample. The PP sample will be further defined in the statistical analysis plan 
(SAP) before database lock. Major efficacy endpoints will be analyzed with the PP sample in 
addition to the ITT sample. 
 

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12.5.2 Description of Baseline Characteristics and Demographics 
Summary descriptive statistics for demographic and baseline characteristics will be provided 
for all enrolled participants. Demographic characteristics will include age, race, sex, body 
weight, and height.  Continuous demographic and baseline data (e.g., age, body weight, and 
height) will be summarized in the electronic medical record. 
 
12.5.3 Medical History 
Medications will be listed in the subject’s electronic medical record. 
 
11.5.4 Use of Medications 
All medications used will be coded using the WHO drug dictionary. The number and 
percentage of participants receiving concomitant medications/therapies will be presented. 
 
12.5.5 Study Completion 
The percent of participants who fail to complete the study, losses to follow-up, times to lost 
to follow up, and reasons for discontinuation (adverse events, other) will be presented. 
 
12.5.6 Efficacy 
All efficacy endpoints will be evaluated on an ITT basis. Major efficacy endpoints will also 
be analyzed using the PP sample to provide supportive or exploratory information. 
 
12.5.7 Primary Efficacy Analysis 
The primary endpoint, namely, patient and graft survival at one year posttransplant, will be 
descriptively summarized with 95% confidence intervals. 
 
12.5.8 Secondary Efficacy Analyses 
The secondary efficacy endpoints will be descriptively summarized with 95% confidence 
intervals (as appropriate). 
 
12.5.9 Safety 
Participants comprising the Safety sample will be included in all safety analyses. 
All adverse events, including posttransplant infections, malignancies, thromboembolic 
events, morbidity, and side effects associated with the low dose steroid-free maintenance 
immunosuppression, will be classified by body system and preferred term according to the 
Medical Dictionary for Regulatory Activities (MedDRA). 
 
Frequency tables by category of event (e.g., serious, related to study therapy, causing the 
discontinuation of study therapy) and by NCI CTC grade will be presented by treatment 
group. The safety displays will include summaries of the occurrence and incidence of 
posttransplant infections, malignancies (including PTLD), thromboembolic events, 
hyperlipidemia, leukopenia, thrombocytopenia, GI toxicity, and cytokine release by 
treatment group. Descriptive statistics (mean, standard deviation, mean change from 
baseline) of laboratory values and vital signs will be presented by treatment group. 
Laboratory values will also be summarized by mean, standard deviation, and change from 
baseline by treatment group. 
 

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12.6 
Planned Interim Analyses 
Enrollment in the trial will be suspended for safety reasons i.e. unexplained adverse events.  
The DSMB will review any events as requested by the Protocol Chair or the medical 
monitor.  They will review listings of all adverse events and laboratory findings periodically.  
  
12.7 
Procedures for Reporting Deviations from the Original Plan 
Any changes in these principal features would require a protocol or SAP amendment, which 
would then be subject to review by the independent Data and Safety Monitoring Board 
(DSMB), study sponsor(s), and regulatory agencies. These changes will be described in the 
final report as appropriate. 
 
13 
Access to Source Data / Documents  
The investigational site participating in this study will maintain the highest degree of 
confidentiality permitted for the clinical and research information obtained from participants 
and donors participating in this clinical trial.  Medical and research records should be 
maintained at each site in the strictest confidence.  However, as a part of the quality 
assurance and legal responsibilities of an investigation, the investigational site must permit 
authorized representatives of the sponsor(s) and regulatory agencies to examine (and when 
required by applicable law, to copy) clinical records for the purposes of quality assurance 
reviews, audits and evaluation of the study safety and progress.  Unless required by the laws 
permitting copying of records, only the coded identity associated with documents or other 
participant data may be copied (obscuring any personally identifying information).  
Authorized representatives as noted above are bound to maintain the strict confidentiality of 
medical and research information that may be linked to identified individuals.  The 
investigational site will normally be notified in advance of auditing visits. 
 
14 
Quality Control and Quality Assurance 
The Investigator is required to keep accurate records to ensure the conduct of the study is 
fully documented.  The Investigator is required to ensure that all case report forms are legibly 
completed for every participant entered in the trial. 
 
The sponsor is responsible for regular inspection of the conduct of the trial, for verifying 
adherence to the protocol, and for confirming the completeness, consistency and accuracy of 
all documented data. 
 
To ensure the reliability of the data recorded in the database, double data entry will be used 
for all fields on the CRF.  The data will be verified by a series of computerized edit checks, 
and all relevant data queries will be resolved on an ongoing basis. When the CRFs are 
complete, they will be reviewed and signed by the Investigator.  All data from the original 
signed CRF will be entered in the database, and a comparison program will be run again. All 
discrepancies will be reviewed, and any resulting queries will be resolved with the 
Investigator and amended on the database.  All elements of data entry (i.e., time, date, 
verbatim text and the person performing the data entry) are recorded in an electronic audit 
trail to allow all data changes in the database to be monitored and maintained in accordance 
with federal regulations. 
 

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15 
Ethical Considerations and Compliance with Good Clinical Practice 
15.1 
Statement of Compliance 
This trial will be conducted in compliance with the protocol, current Good Clinical Practices 
(GCP), adopting the principles of the Declaration of Helsinki, and all applicable regulatory 
requirements. 
Prior to study initiation, the protocol and the informed consent documents will be reviewed 
and approved by an appropriate ethics review committee or Institutional Review Board 
(IRB).  Any amendments to the protocol or consent materials must also be approved before 
they are implemented. 
 
15.2 
Informed Consent 
The informed consent form is a means of providing information regarding the trial to a 
prospective participant and allows for an informed decision about participation in the study.  
All participants (or their legally acceptable representative) must read, sign and date a consent 
form prior to participation in the study, taking study drug and/or undergoing any study-
specific procedures.  If a participant does not speak and read English, the consent materials 
must be translated into a language understandable to the subject. 
The informed consent form must be updated or revised whenever important new safety 
information is available, whenever the protocol is amended, and/or whenever any new 
information becomes available that may affect a patients’ participation in the trial. 
A copy of the informed consent will be given to a prospective participant for review.  The 
Principal Investigator, Co-Principal Investigator, Sub-Investigator and/or research nurse, will 
review the consent and answer questions. The participant will be informed that their 
participation is voluntary and they may withdraw from the study at any time, for any reason, 
without prejudice to their ongoing care. 
 
15.3 
Privacy and Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization.  For subjects that have revoked authorization to collect or use PHI, 
attempts will be made to obtain permission to collect at least vital status (i.e. that the subject 
is alive) at the end of their scheduled study period. 
 
 

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16 
References 
1. 
Ciancio G, Miller J, Garcia-Morales RO, Carreno M, Burke GW 3rd, Roth D, Kupin W, 
Tzakis AG, Ricordi C, Rosen A, Fuller L, Esquenazi V. Six-year clinical effect of 
donor bone marrow infusions in renal transplant patients. Transplantation. 2001; 
71(7):827-35.  
2. 
Ciancio G, Burke GW, Garcia-Morales R, Suzart K, Rosen A, Ricordi C, Kenyon NS, 
Mathew JM, Tzakis AG, Esquenazi V, Miller J.  Effect of living-related donor bone 
marrow infusion on chimerism and in vitro immunoregulatory activity in kidney 
transplant recipients. Transplantation. 2002; 74(4):488-96. 
3. 
Waldmann H, Polliak A, Hale G, et al. Elimination of graft versus host disease by in 
vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human 
lymphocyte antibody (CAMPATH-1). Lancet. 1984; 2(8401):483. 
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Naparstek E, Delukina, M, Or R, et al. Engraftment of marrow allografts treated with 
Campath-1 monoclonal antibodies. Exp Hematol. 1999; 27(7):1210. 
5. 
Novitsky N, Thomas V, Hale G, Waldmann H. Ex vivo depletion of T cells from bone 
marrow grafts with CAMPATH-1 in acute leukemia: graft versus host disease and 
graft-versus-leukemia effect. Transplantation. 1999; 67(4):620. 
6. 
Cull GM, Haynes AP, Byrne JL, et al. Preliminary experience of allogeneic stem cell 
transplantation for lymphoproliferative disorders using BEAM-CAMPATH 
conditioning: an effective regimen with low procedure-related toxicity. Br J 
Haematol. 2000; 108(4):754. 
7. 
Hale G, Jacobs P, Wood L, et al. CD52 antibodies for prevention of graft versus host 
disease and graft rejection following transplantation of allgeneic peripheral blood 
stem cells. Bone Marrow Transplantation. 2000; 26(1):69. 
8. 
Kottaridis PD, Milligan DW, Chopra R, et al. In vivo CAMPATH-IH prevents graft 
versus host disease following nonmyeloablative stem cell transplantation. Blood. 
2000; 96(7):2419. 
9. 
Lockwood CM, Thiru S, Isaacs JD, Hale G, Waldmann H, Long-term remission of 
intractable systemic vasculitis with monoclonal antibody therapy. Lancet. 1993; 
341(8861):1620. 
10. Coles AJ, Wing MJ, Compston DA. Disease activity and the immune set in multiple 
sclerosis: blood markers for immunotherapy. Mult Scler. 1998; 4(3):232. 
11. Killick SB, Marsh JC, Hale G, Waldmann H, Kelly SJ, Gordon-Smith EC. Sustained 
remission of severe resistant autoimmune neutropenia with Campath-1H. Br J 
Haematol. 1997; 97(2):306. 
12. Lim SH, Hale G, Marcus RE, Waldmann H, Baglin TP. CAMPATH-1H monoclonal 
antibody therapy in severe refractory autoimmune thrombocytopenic purpura. Br J 
Haematol. 1993; 84(3):542. 
13. Spitzer TR, Delmonico F, Tolkoff-Rubin N, McAfee S, Sackstein R, Saidman S, Colby 
C, Sykes M, Sachs DH, Cosimi AB. Combined histocompatibility leukocyte antigen-
matched donor bone marrow and renal transplantation for multiple myeloma with end 
stage renal disease: the induction of allograft tolerance through mixed 
lymphohematopoietic chimerism. Transplantation. 1999; 68(4):480-4. 
14. Watson CJ, Bradley JA, Friend PJ, Firth J. Taylor CJ, Bradley JR, Smit S, Jamieson 
NV, Hale G, Waldmann H, Calne R.  Alemtuzumab (CAMPATH 1H) induction 
therapy in cadaveric kidney transplantation—efficacy and safety at five years.  
American Journal of Transplantation.  2005,  5: 1347-53. 

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version date: 25 Sep 2020 
Amendment 6.2 
PI:  Joseph Leventhal 
 
 
 
Page 38                                      Division 
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Transplantation 
 
 
 
 
15. Kirk AD, Swanson SJ, Mannon RB, Batty DS, Bernstein W, Brettman L, Chamberlain 
C, DiMercurio BS, Hunter K, Kampen R, Kleiner D, Tadaki DK, Harlan DM.  
Preliminary results from a human tolerance trial using Campath-1H.  Am J Transplant 
1(sup1). 2001; 136. 
16. Knechtle SJ, Pirsch JD, H Feschner J, Becker BN, Fridela, et al. Campath-1H induction 
plus rapamycin monotherapy for renal transplantation: results of a pilot study. Am J 
Trans. 2003; 3(6):722-730. 
17. Stuart FP, et al. Alemtuzumab facilitates prednisone-free immunosuppression in kidney 
transplant recipients with no early rejection.  Am Trans Cong. 2002: Abstract 1030. 
18. Dixon B. Kaufman, Joseph R. Leventhal, David Axelrod, Lorenzo G. Gallon, Michele 
A. Parker, Frank P. Stuart (2005).    Alemtuzumab Induction and Prednisone-Free 
Maintenance Immunotherapy in Kidney Transplantation: Comparison with 
Basiliximab Induction—Long-Term Results.  American Journal of Transplantation.  
2005, 5 (10): 2539–2548. 
19. Ciancio G, Burke GW, Gaynor JJ, Mattiazzi A, Roohipour R, Carreno MR, Roth D, 
Ruiz P, Kupin W, Rosen A, Esquenazi V, Tzakis AG, Miller J.  The use of Campath-
1H as induction therapy in renal transplantation: preliminary results.  Transplantation 
2004 Aug 15; 78(3): 426-433. 
20. Ciancio G, Burke GW, Gaynor JJ, Carreno MR, Cirocco RE, Mathew JM, Mattiazzi A, 
Cordovilla T, Roth D, Kupin W, Rosen A, Esquenazi V, Tzakis AG, Miller J.  A 
randomized trial of three renal transplant induction antibodies: early comparison of 
tacrolimus, MMF and steroid dosing, and newer immuno-monitoring.  
Transplantation, In press, 2005. 
21. Jin Y, Fuller L, Rosen A, Ciancio G, Burke GW 3rd, Ricordi C, Tzakis AG, Miller J, 
Esquenazi V.  Campath-1H does not alter bone marrow cell regulatory function.  
Hum Immunol, Submitted Feb., 2005. 
22. Mathew JM, Garcia-Morales R, Fuller L, Rosen A, Ciancio G, Burke GW, Carreno M, 
Temple D, Tzakis AG, Ricordi C, Miller J, Esquenazi V.  Donor bone marrow-
derived chimeric cells present in renal transplant recipients infused with donor 
marrow. I. Potent regulators of recipient antidonor immune responses.  
Transplantation. 2000; 70(12):1675-82. 
23. Mathew JM, Carreno M, Fuller L, Ricordi C, Kenyon N, Tzakis AG, Miller J, 
Esquenazi V.  Immunogenicity of cadaver donor bone marrow cells used for the 
induction of allograft acceptance in clinical transplantation.  Transplantation. 1999; 
68(8):1172-80. 
24. Mathew JM, Fuller L, Carreno M, Garcia-Morales R, Burke GW, Ricordi C, Esquenazi 
V, Tzakis AG, Miller J. Involvement of multiple subpopulations of human bone 
marrow cells in the regulation of allogeneic cellular immune responses. 
Transplantation. 2000; 70(12):1752-1760. 
25. Mathew JM, Carreno M, Fuller L, Burke GW 3rd, Ciancio G, Ricordi C, Tzakis AG, 
Esquenazi V, Miller J. Regulation of alloimmune responses (GvH reactions) in vitro 
by autologous donor bone marrow cell preparation used in clinical organ 
transplantation.  Transplantation. 2002; 74(6):846-55. 
26. Jin Y, Fuller L, Carreno M, Esquenazi V, Blomberg BB, Wei YT, Ciancio G, Burke 
GW, Tzakis A, Ricordi C, Millar J. Functional and phenotypic properties of 
peripheral T cells anergized by autologous CD3+ depleted bone marrow cells.  Hum 
Immunol.2002; 63(7):567. 

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version date: 25 Sep 2020 
Amendment 6.2 
PI:  Joseph Leventhal 
 
 
 
Page 39                                      Division 
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Transplantation 
 
 
 
 
27. Jin Y, Fuller L, Esquenazi V, Blomberg BB, Rosen A, Tzakis AG, Ricordi C, Miller J.  
Bone marrow cells inhibit the generation of autologous EBV-specific CTL.  Hum 
Immunol. 2000; 61(6):538-47. 
28. Jin Y, Fuller L, Wei Y, Blomberg BB, Miller J, Esquenazi V.  Bone marrow cells 
promote TH2 polarization and inhibit virus-specific CTL generation.  Hum Immuol. 
2000; 61:1233-41. 
29. Carreno MR, Fuller L, Mathew JM, Ciancio G, Burke GW, Esquenazi V, Ricordi C, 
Tzakis AG, Miller J.  Human donor bone marrow cells induce in vitro “suppressor T 
cells” that functionally suppress autologous B cells.  Hum Immunol. 2003; 64(1):21-
30. 
30. Mathew JM, Blomberg B, Fuller L, Burke GW, Ciancio G, Ricordi C, Tzakis AG, 
Esquenazi V, Miller J.  A novel micro-cell-mediated lympholytic assay for the 
evaluation of regulatory cells in human alloreactive CTL responses.  J Immunol Meth. 
2003; 272(1-2):67-80. 
31. Miller J, Mathew J, Garcia-Morales R, Zucker KE, Carreno M, Jin Y, Fuller L, Burke 
GW, Ciancio G, Tzakis AG, Ricordi C, Olson L, Rosen A, Roth D, Esquenazi V.  
The human bone marrow as an immunoregulatory organ.  Transplantation. 1999; 
68(8):1079-90. 
32. Mathew JM, Miller J.  Immunoregulatory role of chimerism in clinical organ transplan-
tation.  Bone Marrow Transplant. 2001; 28(2):115-9. 
33. Brodignon C, Keever CA, Small TN, et al. Graft failure after T cell depleted human 
leukocyte antigen identical marrow transplants for leukemia: II. In vitro analysis of 
host effector mechanisms. Blood. 1989; 74:2237-2243. 
34. Davies SM, Shu XO, Blazar BR, et al. Unrelated donor bone marrow transplantation: 
influence of HLA A and B incompatibility on outcome. Blood. 1995; 86:1636-1642. 
35. Urbano-Ispizua A, Rozman C, Pimentel P, et al. The number of donor CD3+ cells is the 
most important factor for graft failure after allogeneric transplantation of CD34+ 
selected cells from peripheral blood from HLA-identical siblings. Blood. 2001; 
97:383-387. 
36. Anderson KC, Soiffer R, DeLage R, et al. T cell depleted autologous bone marrow 
transplantation therapy: analysis of immune deficiency and late complications. Blood. 
1990; 76:235-244. 
37. Cornelissen JJ, Van der Holt B, Petersen EJ, et al. A randomized multicenter 
comparison of CD34+ selected progenitor cells from blood vs. from bone marrow in 
recipients of HLA-identical allogeneic transplants for hematological malignancies. 
Exp Hematol. 2003; 31:855-864. 
38. Buckley RH, Schiff SE, Schiff RI, et al. Hematopoietic stem cell transplantation for the 
treatment of severe combined immunodeficiency. New Eng J Med. 1999; 340:508-
516. 
39. Campath-1H [package insert]. Cambridge, MA: Millenium and ILEX Partners, LP; 
2001. 
40. Calne R, Moffatt SD, Friend PJ, et alAopaoo. Campath-1H allows low-dose 
cyclosporine monotherapy in 31 cadaveric renal allograft recipients. Transplantation. 
1999; 68:1613-1616. 
41. Garcia-Morales R, Carreno M, Mathew J, Cirocco R, Zucker K, Ciancio G, Burke G, 
Roth D, Temple D, Fuller L, Esquenazi V, Eskind L, Kenyon NS, Ricordi C, Tzakis 
A, Miller J.  Continuing observations on the regulatory effects of donor-specific bone 

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Amendment 6.2 
PI:  Joseph Leventhal 
 
 
 
Page 40                                      Division 
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Organ 
Transplantation 
 
 
 
 
marrow cell infusions and chimerism in kidney transplant recipients.  
Transplantation. 1998; 65(7):956-65. 
42. Garcia-Morales R, Esquenazi V, Zucker K, Gomez CI, Fuller L, Carreno M, Cirocco R, 
Alamo A, Karatzas T, Burke GW 3rd, Ciancio G, Temple D, Fernandez H, Ricordi C, 
Tzakis A, Miller J. An assessment of the effects of cadaver donor bone marrow on 
kidney allograft recipient blood cell chimerism by a novel technique combining PCR 
and flow cytometry. Transplantation. 1996; 62(8):1149-60. 
43. Garcia-Morales R, Carreno M, Mathew J, Zucker K, Cirocco R, Ciancio G, Burke G, 
Roth D, Temple D, Rosen A, Fuller L, Esquenazi V, Karatzas T, Ricordi C, Tzakis A, 
Miller J. The effects of chimeric cells following donor bone marrow infusions as 
detected by PCR-flow assays in kidney transplant recipients. J Clin Invest. 1997; 
99(5):1118-29. 
44. Heeger PS, Greenspan NS, Kuhlenschmidt S, Dejelo C, Hricik DE, Schulak JA, Tary-
Lehmann M. Pretransplant frequency of donor-specific, IFN-gamma-producing 
lymphocytes is a manifestation of immunologic memory and correlates with the risk 
of posttransplant rejection episodes. J Immunol. 1999; 163(4)2267-75. 
45. Honey K, Cobbold SP, Waldmann H. CD40 ligand blockade induces CD4+ T cell 
tolerance and linked suppression. J Immunol. 1999; 163(9):4805-10. 
46. Nocera A, Cosimi AB, Colvin RB, Gesner ML, Fuller TC. Phenotypic and functional 
analysis of kidney infiltrating T cells escaping monoclonal antibody treatment in 
primate renal transplantation. Transplant Proc. 1988; 20(2):205-6. 
47. Cirocco R.  Cytokines and Th subsets in tolerance.  Curr Op Transplant. 2002; 7:13-7. 
48. Cirocco R, Markou M, Zucker K, Karatzas T, Garcia R, Carreno M, Ricordi C, Burke 
G, Tzakis A, Miller J.  Chimeric quantitation of donor mismatched class II alleles by 
a nonradioactive sequence specific priming assay.  Hum Immunol.  1995; 44(suppl 
1):144. 
49. Snell GD, Dausset J, Nathenson SG. Histocompatibility. New York: Academic Press, 
1976. 
50. *Jin Y, Fuller l, Esquenazi V, Blomberg BB, Burke GW 3rd, Ciancio G, Tzakis AG, 
Ricordi C, Miller J.  Induction of atuto-reactive regulatory T cells by stimulation with 
immature autogologous dendritic cells.  Immunol Invest 2007; 36(2):213-232. 
51. *Cirocco RE, Mathew JM, Burke GW 3rd, Esquenazi V, Miller J.  Killer cell 
immunoglobulin-like receptor polymorphisms in HLA-identical kidney transplant 
recipients: lack of 2DL2 and 2DS2 may be associated with poor graft function.  
Tissue Antigens 2007 Apr; 69 Auppl 1:123-124. 
52. *Jin Y, Hernandez A, Fuller L, Rosen A, Cirocco R, Esquenazi V, Ciancio G, Burke 
GW, Miller J.  A novel approach to detect donor/recipient immune responses between 
HLA-identical pairs.  Hum Immunol 2007 May; 68(5):350-361.   
       
  *additional relevant immune assays conducted by the PI that may be applicable but have not 
been described in detail in the text.  

Protocol 
 
Page 41 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
Appendix 1: Schedule of Events for Years 1 through 3 
HLA Identical Sibling Renal Transplant Tolerance with Donor Hematopoietic Stem Cells and Campath-1H. 
Schedule of Events 
Test 
Day 
Week 
Month 
Timing of Study 
Participation 
00 
0(
Tx
) 
1 
1 
3 
4 
6 
2 
3 
5 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
Visit 
00 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
1
0 
11 
12 
13 
14 
15 
16.1. 16.1. 16.1. 16.1.
GENERAL ASSESSMENTS 
History & Physical, 
Inclusion/Exclusion 
criteria, Informed 
consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chest X-ray and 
ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam! 
X 
 
 
 
 
X 
 
X 
X 
 
X 
X X 
X 
X 
 
X 
 
X 
 
X 
Vital signs! 
X 
 
X 
 
 
X 
 
X 
X 
 
X 
X X 
X 
X 
 
X 
 
X 
 
X 
 QuantiFeron Gold 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse Event 
Assessment 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
Graft Survival 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
Kidney Biopsy 
 
X 
 
 
 
 
 
 
 
 
 
 
x 
 
X1 
 
X2 
 
 
 
X 
LABORATORY ASSESSMENTS 
Blood type/screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HLA Typing 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PRA 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lymphocytoxic cross 
match 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy (serum b-
HCG for females) 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol 
 
Page 42 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
HLA Identical Sibling Renal Transplant Tolerance with Donor Hematopoietic Stem Cells and Campath-1H. 
Schedule of Events 
Test 
Day 
Week 
Month 
Timing of Study 
Participation 
00 
0(
Tx
) 
1 
1 
3 
4 
6 
2 
3 
5 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
Visit 
00 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
1
0 
11 
12 
13 
14 
15 
16.1. 16.1. 16.1. 16.1.
CBC (including 
differential)! 
X 
 
X 
 
 
X 
 
X 
X 
 
X 
X X3 
X 
X 
X 
X4 
X 
X 
X 
X 
Chemistries! 
X 
 
X 
 
 
X 
 
X 
X 
 
X 
X X3 
X 
X 
X 
X4 
X 
X 
X 
X 
Lipids (Chol, Trig, 
HDL, LDL) 
 
 
 
 
 
 
 
 
X 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis! 
 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
Serology-CMV, 
EBV, Hep B, Hep C, 
& HIV (participant 
and donor) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Quantitative PCR for 
CMV reactivation (if 
needed for safety) 
 
 
 
 
 
X 
 
X 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
GFR 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
CD52 (T cells) 
X 
 
X5 
X6 
 
X7 
X7 
X7 
X8 
X8 
X8 
X
8 
X8 
X8 
X8 
X8 
X8 
X8 
X8 
X8 
X8 
Tacrolimus trough 
blood levels* 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Sirolimus trough 
blood levels** 
 
 
 
 
 
 
 
X 
X 
X 
X 
X X 
X 
X 
 
 
 
 
 
 

Protocol 
 
Page 43 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
HLA Identical Sibling Renal Transplant Tolerance with Donor Hematopoietic Stem Cells and Campath-1H. 
Schedule of Events 
Test 
Day 
Week 
Month 
Timing of Study 
Participation 
00 
0(
Tx
) 
1 
1 
3 
4 
6 
2 
3 
5 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
Visit 
00 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
1
0 
11 
12 
13 
14 
15 
16.1. 16.1. 16.1. 16.1.
Donor Renal 
Chemistries and 
Immune Assays 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
MEDICATIONS AND DHSC INFUSIONS 
Campath-1H  
 
X 
 
Day 
4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DHSC infusion #1 
(frozen) 
 
 
 
Day 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DHSC infusion #2 
(fresh) 
 
 
 
 
 
 
 
Month 2-
3 
 
 
 
 
 
 
 
 
 
 
 
 
DHSC infusion #3 
(frozen)*** 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
DHSC infusion #4 
(frozen)*** 
 
 
 
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
Tacrolimus 
 
 
X 
X 
X 
X 
X 
X? 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sirolimus 
 
 
 
 
 
 
 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
 
 
 
 
MMF 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
 
 
 
 
 
 
Valganciclovir 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
 
 
 
 
 
 
 
 
TMP/Sulfa 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
 
 
 
 

Protocol 
 
Page 44 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
HLA Identical Sibling Renal Transplant Tolerance with Donor Hematopoietic Stem Cells and Campath-1H. 
Schedule of Events 
Test 
Day 
Week 
Month 
Timing of Study 
Participation 
00 
0(
Tx
) 
1 
1 
3 
4 
6 
2 
3 
5 
6 
9 
12 
15 
18 
21 
24 
27 
30 
33 
36 
Visit 
00 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
1
0 
11 
12 
13 
14 
15 
16.1. 16.1. 16.1. 16.1.
Clotrimazole or oral 
nystatin 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
 
 
 
 
   *Tacrolimus blood levels are actually to be performed weekly and recorded in the electronic medical record with each clinic visit.  This will 
include the visits as per our standard of care until the   
     drug is discontinued.   
 **Sirolimus blood levels are actually to be performed weekly and recorded in the electronic medical record until month 5 and then monthly as is 
our standard of care until the drug is discontinued. 
***Note: Infusions #3 and #4 will follow similar CRF format. 
TOLERANCE ASSAY STUDIES (SITE PERFORMED) 
Sequential 
measurement of 
regulatory cells and 
their products11, 12, 13, 
14 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
1 Two weeks prior to sirolimus withdrawal. 
2 Two weeks prior to MMF withdrawal. 
3 CBC and blood chemistries will occur weekly until sirolimus is completely withdrawn, then every other week for the first 6 weeks after complete 
withdrawal of sirolimus, then monthly thereafter.   
4 CBC and blood chemistries will occur weekly until MMF is completely withdrawn, then every other week for the first 6 weeks after complete 
withdrawal of MMF, then monthly thereafter. 
5 CD52 QD x 7 days. 
6 CD52 2x/week x 30 days. 
7 CD52 Q/week x 60 days. 
8 CD52 Q2/week x 60 days. 

Protocol 
 
Page 45 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
11 Ex vivo immune assessment will occur for peripheral blood at 6, 18, 30, 36, (visits 42, and 54 months will be captured at the site level not on 
CRF’s). 
12 Ex vivo immune assessment will occur for iliac crest bone marrow at 12, 24, 36, (visits 48, and 60 months will be captured at the site level not on 
CRF’s). 
13 Protocol biopsies will be obtained 2 weeks prior to commencement of sirolimus withdrawal, at 2 weeks prior to MMF withdrawal and 6 months 
after completion of MMF withdrawal.  “For-cause” biopsies will also be obtained in the event of a 20% increase in serum creatinine that is 
unexplained and unresolved within 24 hours. 
14 If the recipient demonstrates donor-specific unresponsiveness in the ELISPOT and micro-CML assays, limiting dilution analysis for the 
enumeration of CTLp will be performed.  This also will include trans-vivo DTH reactions and specialized donor specific antibody assays.Note:  All 
adverse events (X) are to be recorded on case report forms (clinically significant or non-significant). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol 
 
Page 46 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as 
in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
APPENDIX II 
16.1.6 Isolation of CD34-Positive HPC 
 
Growth factor mobilized (G-CSF) donor peripheral blood progenitor cell (PBPC) products will be 
collected by apheresis.  Depending upon the success of the mobilization, a second PBPC collection may 
be required to obtain sufficient PBPCs to perform the CD34+ cell selection step to recover sufficient 
donor CD34+ target cells. 
 
CD34+ cell selection of the mobilized PBPC collection or iliac crest bone marrow will be performed on 
the Miltenyi Biotec CliniMACS selection device.  The CliniMACS device features an automated, 
computer-driven program utilizing a closed system (sterile pathway) with a sterile, disposable plastic 
processing set and sterile buffer solutions for ease of cell processing.   The CliniMACS has been 
approved for clinical trials.  Approved materials and reagents that will be used with the CliniMACS 
device are indicated in Table 1 below.  This cell enrichment method results in greater than 60% CD34+ 
cell recovery and approximately 80-90% CD34+ cell purity with a > 3 log depletion of T-cells.  The 
PBPC products will be processed according to the Operator’s Manual for the CliniMACS selection 
device.  Refer to the standard operating procedure (SOP): CTL 2.6 HCT/P Manufacturing: CD34 
Positive Cell Selection using CliniMACS.  The following steps will be performed to select the CD34+ 
cells and deplete the CD3+ cells: 
 
1. Donor PBPCs will be washed by centrifugation with incubation buffer to remove interfering 
platelets from the PBPC product 
 
2. PBPCs will be incubated with the CD34 monoclonal antibody/super-paramagnetic particle 
conjugate to label the CD34+ cells.  Labeled cells will be incubated for 20 minutes at room 
temperature on a rocking platform to facilitate labeling of cells 
 
3. Labeled cells will be washed twice by addition of 10-20 times labeling volume of phosphate 
buffered saline supplemented with 0.5% HSA and 2mM EDTA.  The mixture will be centrifuged 
at 300 x g for 10 minutes at room temperature using a COBE 2991 Cell Washer.  The supernatant 
will be removed and discarded 
 
4. Selection of CD34+ cells is accomplished by immobilizing the CD34+ cell/antibody/bead mixture 
in a magnetic field and, washing the unbound (by-pass cells) CD3+ cells, and other cellular 
elements, from the column containing the magnetically bound CD34+ cells. 
 
5. After the unbound CD3+ T-cells and other unwanted cells have been removed, the CD34+ cells 
will be released from the column by removing the magnetic field surrounding the column. 
 
6. The resulting enriched CD34+ cell product should contain a minimum of 1-2 x 106 CD34+ 
cells/kg recipient weight with < 5 x 104 CD3+ T-cells/kg for bone marrow selected products, and 
> 2 x 106 CD34+ cells/kg recipient weight with < 5 x 104 CD3+ T-cells/kg depending upon the 
unfractionated CD34+ cell content of the HPC-A product. 
 
7. After completion of the CD34+ cell selection step, the CD34+ enriched product will either be 
administered immediately following CD34+ cell analysis or it will be cryopreserved in 10% 

Protocol 
 
Page 47 
Amendment 6.2 
Version Date: 06 Mar 2020 
 
 
! During the drug withdrawal period, as per Sections 4.7.1 and 4.8.1, these tests are to be performed weekly and recorded both on case report forms, as well as 
in the patients’ charts. 
PI: Joseph Leventhal  
 
 
 
 
 
  
 
 
 
 
dimethyl sulfoxide (DMSO) and Normal Saline solution containing a minimum of 20% 
autologous plasma, if storage is required prior to transplantation. 
 
8. The cryopreserved CD34-enriched HPC-A product will be stored in the vapor phase of a liquid 
nitrogen (LN2) freezer until the recipient has received the preparative regimen.  Following 
administration of the conditioning regimen, the CD34-enriched product will be rapidly thawed in 
a 37oC water-bath and the cells will be infused without further manipulation according to Cell 
Therapy Program policies. 
 
 
Table 1 
Miltenyi CliniMACS Approved Materials & Reagents 
 
 
Materials: 
 
CliniMACS Tubing Set (Ref. No. 161-01), or Large Scale Tubing Set (Ref. No. 162-01) 
 
 
Reagents: 
 
CliniMACS PBS/EDTA Buffer, 1 L bags (Ref. No. 700-25) 
 
CliniMACS CD34 Reagent Kit (Ref. No. 171-01)