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St. Jude Children's Research Hospital HAPNK1 Initial version, dated: 10/18/2012 (IRB approved: 03/04/2013) Activated: April 10, 2013 Revision 0.1, dated 03/29/2013 (IRB Approved: 04/23/2013) Activated: 05/02/2013 Revision 0.2, dated 05/02/13 (IRB Approved: 05/08/2013) Activated: 05/31/2013 Amendment 1.0, dated 07/03/13 (IRB Approved: 09/19/13) Activated: 09/25/13 Revision 1.1, dated 10/31/13 (IRB Approved: 1/6/14) Activated: January 14, 2014 Revision 1.2, dated 09/23/14 (IRB Approved: 10/23/14) Activated: November 3, 2014 Amendment 2.0, dated 8/17/15 (IRB Approved: 12/7/15) Activated: December 18, 2015 Amendment 3.0, dated 03/16/16 (IRB Approved:07/18/16) Activated: Aug.23, 2016 Amendment 4.0, dated 01/10/17 (IRB approved: 03/07/17) Activated: April 7, 2017 Un-numbered Revision, dated: 11/17/2017 (IRB Approved: 11/17/2017)
Amendment 5.0, dated 03/13/18 (IRB Approved: 5/8/2018) Activated: May 17, 2018 Revision 5.1, dated 06/13/2018 (IRB Approved: 10/11/2018) Activated: 10/31/2018 HAPLOIDENTICAL DONOR HEMATOPOIETIC PROGENITOR CELL AND NATURAL
KILLER CELL TRANSPLANTATION WITH A TLI BASED CONDITIONING REGIMEN IN
PATIENTS WITH
HEMATOLOGIC MALIGNANCIES
IDE \#15422 Principal Investigator Brandon M. Triplett, MD1 Co-Investigators Julia Hurwitz, PhD5 Ashok Srinivasan, MD1 Ewelina Mamcarz, MD1 Paul Thomas, PhD6 Thomas Merchant, DO, PhD2 William Janssen, PhD1 Amr Qudeimat, MD1 Shane Cross, PharmD4 Aimee Talleur, MD1 Ben Youngblood, PhD6 Anusha Sunkara, MS3 Paulina Velasquez, MD1 Akshay Sharma, MD1 Renee Madden, MD Guolian Kang, PhD 3 Department of Bone Marrow Transplantation and Cellular Therapy1 Division of Radiation Oncology, Department of Radiological Sciences2 Department of Biostatistics3 Department of Pharmaceutical Sciences4 Department of Infectious Diseases5 Department of Immunology6 St. Jude Children's Research Hospital 262 Danny Thomas Place Memphis, TN 38105
(901) 595-3300 This is a confidential research document. No information may be extracted without permission of the principal investigator.
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table of contents

Study Summary 1.0 Objectives……………………………………………...……………………………….. 1  
|     | 1.1                                                    | Primary Objectives…………………………………………………..…………………                                        | 1   |
|-----|--------------------------------------------------------|---------------------------------------------------------------------------------------|-----|
|     | 1.2                                                    | Secondary Objectives……………………………………………….………………….                                       | 1   |
|     | 1.3                                                    | Exploratory Objectives…………………………………………….…………………..                                     | 1   |
| 2.0 | Background and Rationale…………………………..…………………………………      | 1                                                                                     |     |
|     | 2.1                                                    | Overview……………………………………………………………………………….                                               | 1   |
|     | 2.2                                                    | Indications for HCT and need for alternative donors …………………………………..                   | 2   |
|     | 2.3                                                    | Haploidentical HCT……………………………………………………………………..                                        | 2   |
|     | 2.4                                                    | Rationale for immunomagnetic T cell depletion of hematopoietic progenitor cell graft. | 4   |
|     | 2.5                                                    | Rationale for the reduced intensity regimen……………………………………………                          | 7   |
|     | 2.6                                                    | Rationale for the therapeutic NK cell graft…………………………………………….                         | 8   |
|     | 2.7                                                    | Minimal residual disease……………………………………………………………….                                     | 9   |
|     | 2.8                                                    | Rationale for the present study…………………………………………………………                                 | 10  |
|     | 2.9                                                    | Experience with GVHD prior to amendment 2.0 ………………………………………                           | 10  |
| 3.0 | Protocol Eligibility Criteria…………………………………………………………….. | 11                                                                                    |     |
|     | 3.1                                                    | Inclusion criteria for transplant recipients ……………………………………………..                      | 11  |
|     | 3.2                                                    | Inclusion criteria for haploidentical donors ……………………………………………                        | 13  |
| 3.3 | Gender and minorities…………………………………………………………………         | 13                                                                                    |     |
| 4.0 | Treatment Plan…….………………………………………………………………………..         | 14                                                                                    |     |
|     | 4.1                                                    | Preparative regimen………………………………………….………………………..                                       | 14  |
|     | 4.2                                                    | Donor selection………………………………………………………………………..                                          | 16  |
|     | 4.3                                                    | Donor mobilization and graft collections …………………………………………….                           | 17  |
|     | 4.4                                                    | Graft preparations……………………………………………………...………………                                       | 18  |
|     | 4.5                                                    | Additional progenitor cell graft administration ……………………………...………..                   | 20  |
|     | 4.6                                                    | Donor lymphocyte infusions………………………………………………………….                                     | 20  |
|     | 4.7                                                    | Quality assurance of cellular products ………………………………………………..                           | 22  |
| 5.0 | Medication and Device Information………………………………….…………    | 23                                                                                    |     |  
5.1 Medications.…………………….…………………………………..………………..… 23 Revision 5.1, dated: 06/13/2018 IRB Approval date: 10/11/2018 Protocol document date: 09/06/2018  
|      | 5.2                                                         | CliniMACSTM System…………………………….…..………………….…………….                      | 30   |
|------|-------------------------------------------------------------|----------------------------------------------------------------------|------|
| 6.0  | Required Observations and Evaluations…………………………..……………      | 31                                                                   |      |
|      | 6.1                                                         | Pre/peri/post-transplant and long-term evaluations …………………………………….   | 31   |
|      | 6.2                                                         | Long-term follow-up evaluations…………………………..…………………………..              | 31   |
|      | 6.3                                                         | Evaluation for chimerism and engraftment ………………………………………..           | 31   |
|      | 6.4                                                         | Evaluation for immune reconstitution …………………………………….………….            | 32   |
|      | 6.5                                                         | General viral surveillance………………..…………………………………………….                 | 32   |
|      | 6.6                                                         | Minimal residual disease evaluations ………………………………………………….            | 32   |
|      | 6.7                                                         | Research tests on haploidentical donor …………..…………………………………….         | 33   |
| 7.0  | Evaluation Criteria…………………...…………………………………………               | 33                                                                   |      |
|      | 7.1                                                         | Adverse event monitoring……………………………………………………………..                    | 33   |
|      | 7.2                                                         | GVHD diagnosis and grading…………………………………………………………                     | 33   |
|      | 7.3                                                         | Performance Status…………………………………………………………………….                        | 34   |
|      | 7.4                                                         | Hematologic recovery………………………………………………………………….                       | 34   |
|      | 7.5                                                         | Graft failure……………………………………………………………………………..                         | 35   |
|      | 7.6                                                         | Chimerism………………………………………………………………………………                              | 35   |
| 8.0  | Off-Study Criteria………………………………………………………………………               | 35                                                                   |      |
|      | 8.1                                                         | Recipient criteria………………………………………………………………………..                      | 35   |
|      | 8.2                                                         | Additional notes………………………………………………………………………..                        | 35   |
|      | 8.3                                                         | Donor criteria…………………………………………………………………………..                         | 36   |
| 9.0  | Reporting Criteria and Continuing Review…………………………………………….. | 36                                                                   |      |
|      | 9.1                                                         | Reporting Adverse Experiences and Deaths to St. Jude IRB ………………………   | 36   |
|      | 9.2                                                         | Reporting to St. Jude institutional biosafety committee ……………………………… | 38   |
|      | 9.3                                                         | Reporting to FDA………………………………………………………………………                          | 38   |
|      | 9.4                                                         | Reporting to St. Jude office of regulatory affairs ……………………………………..  | 39   |
|      | 9.5                                                         | Continuing review reports……………………………………………………………..                   | 39   |
|      | 9.6                                                         | Data submission to Miltenyi Biotec……………………………………………………               | 39   |
| 9.7  | Reporting to the CIBMTR………………………………………………………………… 39         |                                                                      |      |
| 10.0 | Statistical Considerations………………………………………………….……………...      | 40                                                                   |      |  
10.1 Statistical design and analysis for primary objective and stopping rules ……..………. 40 Revision 5.1, dated: 06/13/2018 IRB Approval date: 10/11/2018 Protocol document date: 09/06/2018  
|      | 10.2                                                               | Statistical analysis for secondary objectives ……………………………….………….   | 45   |
|------|--------------------------------------------------------------------|--------------------------------------------------------------------|------|
|      | 10.3                                                               | Analysis for exploratory objectives…………………………………….………………           | 45   |
| 11.0 | Data Acquisition and Quality Assurance Monitoring ……………..……….…………… | 46                                                                 |      |
|      | 11.1                                                               | Enrollment on study….…………………………………………………………………                     | 46   |
|      | 11.2                                                               | Data submission ……………………………………………………………………….                       | 46   |
|      | 11.3                                                               | Quality assurance monitoring…………………………………………………………..               | 46   |
| 12.0 | Obtaining Informed Consent…………………………………………………………………..              | 47                                                                 |      |
| 13.0 | References………………………………………………………………………………………                        | 48                                                                 |      |
| A    | Appendix A……………………..……………………………………….……………………..                     | 55                                                                 |      |
| B    | Appendix B……………………..……………………………………….……………………..                     | 56                                                                 |      |
| C    | Appendix C……………………..……………………………………….……………………..                     | 64                                                                 |      |
| D    | Appendix D……………………..……………………………………….……………………..                     | 67                                                                 |      |
| E    | Appendix E……………………………………………………………………………………..                       | 68                                                                 |      |
| F    | Appendix F……………………………………………………………………………………..                       | 72                                                                 |      |
| G    | Appendix G……………………………………………………………………………………..                       | 73                                                                 |      |
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protocol title

Haploidentical Donor Hematopoietic Progenitor Cell and Natural Killer Cell Transplantation with a TLI
based Conditioning Regimen in Patients with Hematologic Malignancies
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brief overview study population

Patients with high-risk hematologic malignancies who would likely benefit from allogeneic hematopoietic cell transplantation (HCT). Patients who do not have a suitable HLA matched related or unrelated donor will typically be offered enrollment on one of two alternative donor protocols that will be run in parallel. Those with a suitable HLA matched sibling or unrelated donor identified will be eligible for participation ONLY if the donor is not available in the necessary time.  
In general, patients who have a suitable HLA matched haploidentical donor (matched ≥ 3 of 6), will be preferentially offered enrollment on HAPNK1. If the patient is medically eligible, but there is no suitable KIR mismatched haploidentical donor available, suitable cord blood units are available, and the patient is eligible for an institutional cord blood protocol, then the patient will be prioritized for enrollment on the current institutional cord blood transplant protocol.  
Participant must be less than or equal to 21 years old. Additional eligibility criteria are specified to assure sufficient multi-organ system function.
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intervention brief outline and objectives

In this study, participants with high-risk hematologic malignancies undergoing hematopoietic cell transplantation (HCT), who do not have a suitable human leukocyte antigen (HLA) matched related/sibling donor (MSD) or matched unrelated donor (MUD) identified, will receive a haploidentical donor HCT with additional natural killer (NK) cells.  
The assessments and follow-up evaluations noted in the protocol follow the St. Jude standard operating procedures (SOP) for all recipients of allogeneic HCT.  
The primary objective of the study is to estimate the rate of successful engraftment at day +42 posttransplant in patients undergoing haploidentical HCT plus NK cell transplantation with a TLI based conditioning regimen. Secondary aims will assess overall survival, event-free survival, risk of relapse, graft versus host disease (GVHD), transplant related mortality (TRM), transplant related morbidity, and immune reconstitution.
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intervention brief outline and objectives

CliniMACS CD56+ enrichment -- IDE 15422 IDE Cross-reference:
CliniMACS CD34+ enrichment CliniMACS CD45RA depletion *Criteria for Evaluation - Safety and Efficacy*
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safety

The primary measures of safety will be the rate of therapy related death and the rate of severe graft versus host disease. Ongoing assessment of toxicity will be done using the NCI CTCAE version 3.0 Acute and chronic graft-vs.-host disease will be evaluated using established staging criteria and expert consensus guidelines.
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efficacy

The primary measure of efficacy will be rate of successful engraftment. Neutrophil and platelet engraftment will be determined using the parameters put forth by the Center for International Blood and Marrow Registry. Assessments will be made upon review of daily complete blood count and serial chimerism studies. Bone marrow studies for disease status evaluation will be performed at approximately 21 days, 100 days, and 1 year post-transplant. Testing will include a research evaluation for minimal residual disease. Immune reconstitution will be determined by standard blood testing of T- and B cell subsets. Research tests required for participation will examine NK and T-cell function and reconstitution.
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data analyses

Anticipated primary completion date: 07/01/2019 Anticipated study completion date: 07/01/2020 Time frame for primary outcome measure: 42 days post-transplant
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data management including statistical evaluations

Protocol compliance, data collection including safety data, and reporting will be carried out by the Department of Bone Marrow Transplantation and Cellular Therapy Research Office. Statistical considerations and ongoing analysis will be conducted by Dr. Guolian Kang and designated associates within the St. Jude Department of Biostatistics.
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human subjects

The risks to participants are primarily related to the cellular infusions and the conditioning regimen.  
The allogeneic stem cells may induce serious and possibly fatal disorders such as GVHD, venoocclusive disorder and post-transplant lymphoproliferative disease. Because of the required conditioning, recipients are at high-risk for serious and possibly life-threatening infection, bleeding, and anemia. Adverse events will be treated, monitored, and reported appropriately. Possible benefits of participation include obtaining and/or sustaining disease remission. In addition, there is the possibility of psychological benefit from knowing participation has helped researchers gain more understanding about the efficacy of haploidentical HCT. Alternatives to participation are identified as chemotherapy and/or irradiation without transplant, research treatment if available, and/or supportive therapy. The possible benefits, alternatives to participation, and side effects, including that there may be unknown side effects of treatment, are detailed in lay language within the respective informed consent document.
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1.0 objectives 1.1 primary objective

1.1.1 To estimate the rate of successful engraftment at day +42 post-transplant in patients who receive haploidentical donor stem cell plus NK cell transplantation with TLI based conditioning regimen for high risk hematologic malignancy.
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1.2 secondary objectives

1.2.1 Estimate the incidence of malignant relapse, event-free survival, and overall survival at one-year post-transplantation.  
1.2.2 Estimate incidence and severity of acute and chronic (GVHD). 1.2.3 Estimate the rate of transplant related mortality (TRM) in the first 100 days after transplantation.
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1.3 exploratory objectives

1.3.1 Assess the relationship between pre-transplant minimal residual disease (MRD) with transplant outcomes.  
1.3.2 Record immune reconstitution parameters, including chimerism analysis, quantitative lymphocyte subsets, T cell receptor excision circle (TREC) analysis, V-beta spectratyping, and lymphocyte phenotype and function.  
1.3.3 Describe the use of CD45RA-depleted DLI for recipients who have severe viral infections, disease recurrence or progression, or poor immune reconstitution. Assess and record efficacy of CD45RA-depleted DLI for these conditions, and all adverse events that are related to CD45RA-depleted DLI.
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2.0 background and rationale 2.1

Allogeneic HCT is a potential curative therapy for various pediatric hematologic malignancies, however approximately 25-60% of eligible pediatric recipients will not have a human leukocyte antigen (HLA)-matched related/sibling donor (MSD) or a HLA
matched unrelated donor (MUD).1-4 The lack of an adequate MUD for a substantial number of patients, and the lengthy duration of the donor search process in those with high risk of relapse, have prompted the search for alternative donors, including haploidentical family donors and unrelated umbilical cord blood (UCB) grafts.3,4 Nearly all patients have a readily available mismatched family member (haploidentical)
donor, and haploidentical HCT is an effective therapy for patients with hematologic malignancies.5,6 In particular, when extensive T-cell depletion was utilized, the presence of KIR mismatch dramatically reduced the risk of relapse in patients who received haploidentical HCT for high-risk leukemia.7-9 This institution has a track record of success using haploidentical donors for patients with hematologic malignancies,10 and we hypothesize that in patients who require an alternative donor HCT, those with a KIR mismatched donor are likely to benefit most from haploidentical HCT. Therefore, those with only KIR matched haploidentical donors, or those without a suitable haploidentical donor, will be prioritized to UCB HCT. However, with the adoptive transfer of large numbers of memory T-cells in this study, NK cell alloreactivity may no longer be required for adequate control of relapse. Therefore, patients without suitable HLA-matched or UCB
options, and only KIR matched haploidentical donors, may enroll on this study. Here, we propose a single institution prospective phase II trial to estimate the efficacy of haploidentical HCT with additional NK cells in pediatric patients with hematologic malignancies.  
This study will evaluate the safety and efficacy of haploidentical donor allogeneic HCT
with additional NK cells for patients with high risk hematologic malignancies, through the estimation of donor engraftment, survival, relapse, acute and chronic GVHD, chimerism, toxicity, disease recurrence, and post-transplant cellular therapies based on chimerism results.
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2.2 indications for hct and

Allogeneic HCT has become a widely accepted curative therapy for many childhood hematologic malignancies that cannot be cured with chemotherapy alone.11-14 Studies at St.  
Jude and other institutions have demonstrated that HCT is potentially curative for patients with CML, AML, ALL and MDS.15-17 Furthermore, patients with non-Hodgkin or Hodgkin lymphoma who recur after an autologous HCT may be successfully treated with allogeneic HCT.18,19 Importantly, the criteria for patients who are suitable for allogeneic HCT (i.e. would be expected to benefit from allogeneic transplantation) change as new outcome data are compiled and published. For example, less than a decade ago, there was a clear advantage for allogeneic HCT for Ph+ ALL in CR1.13 However, the use of tyrosine kinase inhibitors combined with intensive chemotherapy may have abrogated this advantage.20,21 It is imperative that the transplant physician be aware of the latest data and consensus recommendations14,22 when assessing the suitability for allogeneic HCT.  
Unfortunately, only approximately 30% of patients with hematologic malignancies that are suitable for allogeneic HCT will have a suitable MSD and approximately another 30% will have an MUD. 1-4 Thus, a significant number of patients will not have an appropriate MSD
or MUD donor available in a timely manner, and will require an alternative hematopoietic progenitor cell (HPC) source from haploidentical or unrelated UCB donors. Although, alternative donors have been shown to be suitable to treat children with high-risk hematologic malignancies, they may be associated with increase incidences of graft failure, TRM, GVHD, and delayed immune reconstitution when compared to recipients of MSD or MUD.23,24 Novel strategies are still needed to optimize outcomes in this group.
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2.3 haploidentical hct

Haploidentical donors are viable alternative donors since family members are highly motivated, easily accessible, and readily available for most patients. Along with other institutions, St. Jude has shown haploidentical HCT to be an effective therapy for patients with hematologic malignancies.5,6,25-27 Due to the high potential for GVHD with the degree of HLA mismatch seen in haploidentical HCT, haploidentical grafts are often extensively T-cell depleted prior to infusion. Early results with T-cell depleted haploidentical HCT
were disappointing due very high rates of graft failure and infections.28 Significant progress was achieved with the use of mega-dose of T-cell depleted HPC after highintensity conditioning.26 Aversa and colleagues reported successful long-term engraftment with minimal GVHD in 43 patients with leukemia (including 15 children) using high doses of CD34+ enriched haploidentical cells, purified by positive (CD34+) and negative (CD3lectin agglutination) selection.29,30 Utilizing haploidentical grafts and megadose cell therapy (>1 x 107 CD34+ cells/kg), Handgretinger and colleagues reported similar results in 23 pediatric patients. Prompt engraftment with rapid immune reconstitution was associated with a low incidence of GVHD, even without the use of pharmacologic GVHD
prophylaxis.31 Building on the initial studies above, St. Jude has gained considerable expertise with haploidentical HCT in children.  
8-10,32-36 Over the past decade (2001-2010), more than 220 mismatched related donor HCT have been performed at St. Jude. Initially, most haploidentical HCT performed in this institution were for patients with relapsed and refractory hematologic malignancies, however as it became a more successful therapeutic maneuver, it has become a primary transplant option for patients who lack a well matched donor.10 Recent studies suggest that alloreactive NK cells play a role in graft-vs.-leukemia (GVL)
and influence outcomes of patients with hematologic malignancies after haploidentical HCT.7,3742 In addition to the anti-leukemia effects, NK cells are capable of reducing the incidence of graft rejection and GVHD in animal models; the former is in part based on the mechanism of donor NK cells against patient's T cells, and the latter on donor NK cells against patient's antigen presenting cells.7,40 Taken together, the presence of alloreactive donor NK cells may have multiple advantageous effects, including reduction in (1) relapse (2) graft rejection (3)
GVHD and (4) viral infections.43 The NK cell alloreactivity depends on the balance of signals mediated through activating and inhibitory KIRs on the NK cell. KIRs recognize their ligands, HLA Class I molecules on recipient antigen presenting cells. Some of the inhibitory KIRs recognize specific HLA class I ligands including the well-defined specificity of KIR2DL2/3 for the HLA-Cw group-1 epitope, the specificity of 2DL1 for the Cw group-2 epitope, and the specificity of 3DL1 for the HLA Bw4 epitope. Determination of both donor KIR and recipient HLA typing, allows categorization of KIR match versus mismatch. KIR genes segregate independently of HLA genes, such that matching for HLA does not confirm KIR matching.  
In patients who received extensively T-cell depleted haploidentical HCT for high risk leukemia at St. Jude, the presence of KIR mismatch dramatically reduced the risk of relapse.9 Thus, patients with high risk hematologic malignancies who lack an HLAmatched donor but have a KIR mismatched haploidentical donor available would be expected to benefit the most from the NK cell alloreactivity in haploidentical HCT. Therefore, KIR mismatched donors will be prioritized when available.
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2.4 rationale for immunomagnetic t

Improvements in engraftment were achieved when Handgretinger et al demonstrated that HLA barriers of engraftment in recipients of haploidentical grafts maybe overcome by the infusion of a large number of CD34+ cells (>107 CD34+ cells/kg).26 Collection of megadose CD34+ has become feasible with the implementation of treating donors with G-CSF before donation, which has been shown to increase the number CD34+ cell collected.44 Donor T cells in the haploidentical graft play a major role in mediating GVHD - as few as 3 x 104 Tcells/kg can cause GVHD.27 However, new methods of TCD have been developed to generate a graft with very low T cell content, such that the risk of GVHD is decreased. T cells can be removed from the HPC graft by direct removal of T cells (negative selection)
or selection of CD34+ progenitors (positive selection). The transplantation program at St.  
Jude has had success using either method of T-cell depletion in haploidentical donor transplantation. In this study, CD34+ selection by CliniMACS will be the initial method used for T-cell depletion of the HPC graft. This was used successfully in INFT2 in which the HPC graft was often collected early and frozen until day 0 followed by collection and fresh infusion of an NK cell graft the following week.  
Although positive selection allows for extensive T-cell depletion (TCD), the graft is devoid of other important cell populations such as NK cells and myeloid cells such as dendritic cells and monocytes.45,46 In addition, this extensive degree of TCD has significant negative effects on the time to donor immune competency. Donor T cells are critical for reconstituting the allogeneic host immune system, and lymphocyte recovery is an important determinate of outcome post-transplant.47,48 Transplants that employ TCD result in elimination of most memory T cells, leading to protracted immune dysfunction- an effect that becomes more severe with higher intensity conditioning regimens.32,49 This results in an increased rate of opportunistic infections. Indeed, viral infections are the most common cause of death of children receiving haploidentical transplants.50 The majority of these infections occur within the first 6 months following transplantation, when T cell immunity is the lowest.51 Reconstitution of immunity can be partially restored by therapeutic infusions of donor cytotoxic lymphocytes.47,52-54 However, in addition to requiring significant resources and expertise, the cells must be engineered or selected for specific infections - making this approach impractical for broad application.55 CD45, also called the leukocyte common antigen, is expressed on nearly all hematopoietic cells in various isoforms related to stage of development and activation.56 Among the various isoforms is CD45RA, which selectively identifies naïve T cells.57 Naïve T cells have fully matured but do not proliferate until they encounter the specific ligand that their receptor recognizes.58 If such a proliferative signal occurs after antigen recognition, then there is a marked, antigen-specific cell expansion and inflammatory response. While many of these cells will undergo apoptosis after the initial response, others are rescued from immune retraction and will persist as memory T cells, the majority of which then become CD45RA.59 Having responded to antigen, memory T cells are no longer naïve and are thus capable of more rapidly responding to future infectious challenges.60 Once generated, memory cells persist in the circulation as a diverse cell pool that is critical for long-term infection control.61 A more ideal cell product infusion would offer adoptive transfer of a diverse lymphocyte repertoire capable of effectively rejecting a variety of pathogens, as well as malignant cells. It is hypothesized that the elimination of only CD45RA+ naïve T cells from an infused cell product would not significantly negatively impact memory responses to infection. Among CD4+ T cells, the CD45RA- subset has equivalent "helper" functions as CD45RA+ cells in the generation of alloreactive cytotoxic T cells.62 Further, it is the CD45RA- memory cells that are responsible for aiding B-cell differentiation and antibody production.  
The various strategies thus far reported for selective T cell depletion largely spare CD45RA+ naïve cells, including those that target CD25. Only approximately 5% of naive CD4 cells are CD25+.55 There is potential advantage in targeting naïve T cells for lymphocyte depletion.  
Human studies have shown that sorted donor CD45RA+ naïve T cells are far more alloreactive than all memory subsets tested.63 In animal models, infusions of fractionated memory T cells do not cause physical or histologic evidence of GVHD, independent of Treg numbers. Naïve T cells, conversely, are potent inducers of GVHD, even despite CD25 depletion before infusion.55 Moreover, in similar models, lymphocyte infusions specifically depleted of naïve T cells do not cause GVHD.64 Allografts containing high numbers of CD45RA+ naïve Tregs may be associated with lower risk of acute GVHD in haploidentical transplantation using unmanipulated, fully T cell-replete products.65 The proposed approach will deplete this population - potentially abrogating this effect.  
After showing such promise in animal models, selective CD45RA depletion has begun to be studied in human marrow transplantation, including at this institution. CD45RA depletion by magnetic beads is feasible and effective for naïve T-cell depletion. Data provided by Miltenyi has shown this method to be an effective means of depletion, with greater than 3-fold reduction in number of CD45RA+ cells. Experiments were performed in the Human Applications Laboratory to qualify the CD45RA+ depletion procedures. Hematopoietic progenitor cell products were purchased that were obtained from G-CSF mobilized normal donors by apheresis. The HPC,A products were depleted of CD45RA+ cells following a procedure provided by Miltenyi Biotech. Briefly, the cells were incubated with the CD45RA Microbead reagent followed by washing to remove unbound beads. The labeled cells were then applied to the CliniMACS device (Depletion Tubing Set) and the CD45RA+ cells removed using the Depletion 3.1 program. Flow cytometric analysis before and after depletion was performed following procedures provided by Miltenyi Biotech. The results of the experiments are presented in the table below. An example flow cytometric analysis of CD3+ cells is also included.  
| Experiment # 1              | Experiment #2   |               |                |         |
|-----------------------------|-----------------|---------------|----------------|---------|
| Pre-depletion               | Post-depletion  | Pre-depletion | Post-depletion |         |
| (x 106 )                    | (x 106 )        | (x 106 )      | (x 106 )       |         |
| Nucleated Cells             | 29748.0         | 10270.0       | 26166.0        | 10981.0 |
| CD3+                        | 12238.3         | 4555.8        | 6353.1         | 1985.4  |
| CD3+CD45RA+                 | 7877.3          | 4.1           | 3520.8         | 2.2     |
| CD34+                       | 636.6           | 311.2         | 693.5          | 395.3   |
| CD3+ CD45RA+  Log Depletion | 3.28            | 3.21          |                |         |  
  
For the 26 patients enrolled on this protocol prior to amendment 2.0, there was no maximum naïve T-cell (CD3+CD45RA+) dose. Although there is no correlation with naïve T-cell dose and the occurrence of GVHD so far in this or any other institutional CD45RA-depleted transplant protocol, rare patients have received naïve T-cell doses in excess of 105/kg. In HAPREF, which utilized CD3+ depletion, the conventional T-cell dose cap was raised to 1.5 x 105/kg, and the rate of acute GvHD was noted to be increased. Returning the maximum dose down to 1 x 105 CD3+ cells/kg in HIFLEX again provided an acceptably low rate of acute GVHD. Because approximately ½ of circulating T cells are naïve (and therefore 1 x 105 conventional T cells would contain approximately 0.5 x 105 naïve T-cells), in this trial the naïve T-cell (CD3+CD45RA+) dose in the CD45RAdepleted product will be capped at 0.5 x 105/kg (0.05 x 106) starting with amendment 2.0.  
Of the first 26 patients to receive transplantation and complete one year of follow-up on this protocol, 4 have experienced morphologic relapse. Two of the 4 demonstrated haplotype loss.66 This indicates that the adoptively transferred memory T-cells have antileukemia effects. In this setting then, KIR mismatch driven NK cell alloreactivity may no longer be required for disease control as it was in previous studies utilizing extensively Tcell depleted haploidentical donor transplantation.
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2.5 rationale for the reduced

The immunological effect of graft versus leukemia (GVL) after allogeneic HCT has allowed reduction in the intensity of conditioning as HCT was no longer reliant on the cytotoxicity of high dose of chemotherapy to eradicate disease. Reduced-intensity conditioning (RIC) regimens have the potential to improve the outcomes of allogeneic HCT by decreasing the acute TRM.67-69 Fludarabine-based RIC regimens are safe and effective alternatives to total body irradiation (TBI) in haploidentical HCT.32,70,71 In addition, intensification of immunoablative conditioning using ATG, OKT3, or alemtuzumab, results in the decrease incidence of GVHD and graft rejection as well.71-74 In our first RIC haploidentical HCT protocol REFSCT, we evaluated a RIC regimen for pediatric patients with refractory hematological malignancies or considered at high risk for TRM with a full myeloablative regimen. The RIC regimen consisted of fludarabine
(200mg/m2), thiotepa (10mg/kg), melphalan (120 mg/m2) and Orthoclone-OKT3 and enrolled 25 patients from 2003-2005. Out of the 25 participants, 22 engrafted with a median of 10 days (range, 7-12) and 3 experienced graft failure. The patients with graft failure, one patient had disease relapse and 2 were salvaged with stem cells infusions from their first transplant donor. The median time to platelet engraftment to 20,000/mm3 and 50,000/mm3 was 17 days (range, 12-36) and 17 days (range, 12-76), respectively. The cumulative incidence of overall grade III-IV acute GVHD and chronic GVHD was 8% and 28%, respectively. Of those participants who died, 13 were due to relapsed disease (at a median of 105 days post-HCT). Only 4 (16%) died of TRM, despite most of the patients having received one prior allogeneic HCT - 1 died of GVHD, 1 of infection, 1 of hemorrhage, and 1 of cardiomyopathy. In summary, RIC allows for engraftment with an acceptable rate for graft failure, GVHD, and TRM.  
Two subsequent haploidentical HCT studies at St. Jude (HAPREF and HIFLEX) utilized non-TBI preparative regimens with a fludarabine, thiotepa, melphalan backbone. Twentyseven patients with high risk hematologic malignancies were transplanted on one of these two protocols with OKT3 as in vivo T-cell depletion, followed by infusion of a CD3+
TCD graft. The one-year EFS was 51.9% ± 9.6%. These studies have demonstrated the EFS and TRM in TCD RIC HCT from mismatched haploidentical donor compare favorably with reports from MUD.  
Importantly however, OKT3 was an important component of the preparative regimen for these transplants, and it is no longer commercially available. Our most recent front-line haploidentical donor HCT trial (HIFLEX) has substituted Campath for OKT3. Early results indicate that survival is similar between patients on HIFLEX treated with OKT3 versus Campath (data not shown). However, requirement for the use of therapeutic DLI has risen substantially. Only 1 of the 11 patients who received OKT3 on HIFLEX required more than 2 DLI in the first 100 days post-HCT. In contrast, 9 of the first 14 patients who received Campath on HIFLEX required more than 2 DLI in the first 100 days post-HCT. This is likely due to Campath's anti-NK cell effects, leading to an increased clinical need for early T-cell add back in the form of DLI. Unfortunately, with OKT3's withdrawal from market, there is no commercially available T-cell specific (NK cell sparing) antibody. Therefore, in this protocol, we plan to use total lymphoid irradiation (TLI) in the preparative regimen to provide bidirectional tolerance in substitution of OKT3.  
TLI was initially developed with curative intent in patients with Hodgkin Lymphoma, and it was its use in these patients that led to the discovery of TLI's alteration of T-cell specific immune responses. Years of pre-clinical research on TLI have shown that the use of TLI
promotes engraftment and reduces GvHD.75 This work was then followed up with therapeutic trials in humans with hematologic malignancy, which showed that conditioning with TLI and ATG alone allowed a high rate of durable donor engraftment, a low rate of GvHD, and preservation of evidence of graft versus malignancy effect, in patients who received HLAmatched related or unrelated donor grafts.76,77 We have significant institutional experience using TLI-based regimens for allogeneic transplantation (n=29 as of June 2012). Importantly, TLI-based conditioning has allowed successful salvage haploidentical transplantation in patients who failed previous allo-HCT. Nine patients have received TLI-based conditioning with haploidentical donor HCT after experiencing previous allograft failure (3 had primary graft failure, 4 had initial engraftment with acute rejection, and 2 had late graft failure). The same haploidentical donor was utilized in 5 of the 9 salvage HCT, with 4 patients receiving a new (haploidentical) donor. Eight of 9 patients (89%) experienced durable engraftment at a median of 12 days (range 10 - 27 days). The remaining patient had primary graft failure due to progressive disease. This experience indicates that TLI is effective for facilitation of engraftment, even in patients receiving a mismatched haploidentical donor graft and with a history that indicates a very high risk of graft failure. There is additional published experience from Germany in which TLI - given as a single 7Gy fraction - was utilized in 14 adult and pediatric patients for reconditioning after graft failure/rejection.78 Despite most of the patient having haploidentical donors, engraftment was obtained in all evaluable patients. In addition, TLI was well tolerated in this pediatric population that had recently received another (typically myeloablative) preparative regimen. Finally, 10 patients in Chile received TLI-containing preparative regimen, similar to our proposed regimen, as a part of their haploidentical donor preparative regimen.79 TLI was given as a single dose at 7Gy. Nine out of 10 patients experienced rapid donor engraftment, with relapse as the cause of the one primary graft failure. Six of 10 patients were alive and disease free at one year, 3 died of progressive disease, and one of infection.
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2.6 rationale for the therapeutic

Relapse, infection, and GvHD remain major concerns after HCT. Donor lymphocyte infusion (DLI) containing all types of lymphocytes (B, T, and NK cells) is a viable option for the prevention and/or treatment of both relapse and infection, but conventional
(unfractionated) DLI is limited by the risk of T-cell mediated GVHD.80 Another therapeutic option is the infusion of large number of donor-derived NK cells.43,81-83 In recent years, it has become clear that NK-cell alloreactivity can exert significant effects in allogeneic transplantation. NK-cell alloreactive effects can include a reduction in the rate of rejection, a reduction in GvHD, and a reduction in rate of relapse.7,84 In addition, we and others have shown that patients who have a more robust post-HCT NK-cell recovery demonstrate reduced relapse rates.85,86 Thus, we will provide an infusion of additional NK
cells from the haploidentical donor as a therapeutic cell product of purified NK cells (TCNK).  
Donors on this study will therefore undergo additional leukapheresis on (or around) patient day +5 to obtain the cells for the TC-NK. (See section 4.4). This will be 5 days after the completion of G-CSF mobilization and hematopoietic progenitor cell collection if a fresh stem cell graft is utilized. Donor WBC count is typically returning towards, but still slightly above, baseline at this timepoint.87 Importantly, peripheral blood NK-cell quantities are not significantly altered with G-CSF mobilization and leukapheresis collection in healthy donors.88 In addition, all three patients collected on HAP3R (using this schedule) have met target cell doses for the TC-NK products. The TC-NK will be isolated from the obtained donor mononuclear cells (MNC) in a two-step process. Initially, the MNC will be CD3+ TCD followed by a CD56+ NK enrichment using the CliniMACS
immunomagnetic selection program. TC-NK have been generated using this procedure for the following protocols: NKHEM, INFT2, NKAML, and AML08. The average content of the first 50 TC-NK generated are listed in the table below. We are able to generate a therapeutic cell product that is highly purified for NK cells with minimal contamination of T cells. The final product volume will be approximately 10mL.  
TC-NK Average Standard Deviation **Purity**
NK dose (106 CD56+ cells/kg) 26.6 ± 22.6 96.5%.  
T cell (106CD3+cells) 0.46 ± 0.69 T cell dose (106CD3+ cell/kg) 0.03 ± 0.03 To date, the infusion of TC-NK has been safe and tolerable in both the transplant and cell therapy setting. On INFT2, 13 patients received a TC-NK after haploidentical donor HCT and were evaluable for toxicity. No patient developed an acute infusion reaction to the infusion of TC-NK. One patient had NCI Grade 2 generalized edema the day following the TC-NK, however this is a known and common side effect of allogeneic HCT. Fifty-two patients that have received TC-NK in therapeutic cell therapy protocols were evaluable for toxicity at this writing. (25-NKAML, 19-NKHEM, 8-AML08) Some received TC-NK
following a five-day clofarabine, etoposide, cyclophosphamide regimen, while others received TC-NK after fludarabine, cyclophosphamide therapy. No patient developed an acute infusion reaction. One patient developed hypertension that required a single dose of anti-hypertensive during the night after the infusion.
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2.7 minimal residual disease

Despite the fact that the vast majority of patients with high-risk leukemia are in clinical remission prior to HCT, relapse remains a primary cause of failure after HCT. Detection of minimal residual disease (MRD) and early intervention may improve the clinical outcome. Detection of leukemic cells that are below the limits of detection by standard morphologic examination allow early interventions when the patients are MRD positive but still in remission. By using flow cytometry and polymerase chain reaction (PCR) amplification of antigen receptor genes in tandem, investigators at our institution have been able to conduct MRD studies in 80 consecutive ALL cases.89 Results of St. Jude institutional studies have shown that detection of MRD by immunologic techniques at any point in the treatment course is a powerful predictor of relapse in children with ALL.89-91 However, other studies suggest that eradication of all acute leukemia cells may not be a prerequisite for cure.92,93 Similar to conventional-dose therapy, controversy also exists on the implication of MRD in the setting of HCT. Unlike CML, there is a paucity of data on the natural history of AML and ALL patients who have MRD after HCT, and how pre-transplant MRD levels influence posttransplant outcomes.94,95 It is unclear whether they are also at greater risk of relapse; and whether further pharmacological or immunologic therapy indeed prolongs survival and increases cure rates. Thus, for the participants who are enrolled in this protocol who are unable to proceed to posttransplant immunomodulatory protocols, we will gather the MRD information together with hematopoietic chimerism in a descriptive manner to study the relationship between MRD and chimerism in this large cohort of patients. The knowledge gained from this study should allow their future application to guide therapeutic interventions.
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2.8 rationale for present study

Many patients with high risk hematologic malignancies that would benefit from allogeneic HCT do not have an HLA-matched donor available in the necessary time. This institution has had success with the use of haploidentical donor HCT for such patients. In particular, when a homogenous patient population was examined, outcomes with haploidentical donor HCT appear to be similar to those of conventional allogeneic HCT.10 Thus haploidentical HCT should be offered as a viable option to candidates with high-risk hematologic malignancy that lack a suitable HLA-matched donor, and have a suitable haploidentical donor.
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2.9 experience with gvhd prior

The GVHD syndromes experienced by patients on this protocol differ from conventional Tcell-replete transplantation. Prior to amendment 2.0, 26 patients received naïve Tcelldepleted HCT (range in survivors 180 - 736 days post-HCT). Seven patients developed grade II-IV acute GVHD (1 - II, 4 - III, 2 - IV). GVHD prophylaxis was sirolimus in the first 9 patients, and MMF for the remaining patients. The median duration of GVHD prophylaxis was 18 (range 0 - 154) days. One patient had upper GI symptoms a few months posttransplant in conjunction with a rash and was diagnosed with grade II acute GVHD. The six patients with severe (III-IV) GVHD presented more atypically. One patient initially developed diarrhea with frank blood. Endoscopy showed severe mucosal injury with a broad differential diagnosis, and her prophylactic MMF was stopped. One week later she had fever, an elevated CRP above 20mg/dL, and a new rash. She responded to infliximab and systemic steroids. A second patient developed hepatitis with an HHV6 viral load over 5 log. One week later she developed a rash and the bilirubin continued to rise, therefore systemic steroids were initiated. The third patient presented with fever, nausea, and a rise in liver transaminases followed by increased bilirubin. Diarrhea and rash followed a few days later with a rise in CRP to 15mg/dL. Systemic steroids and infliximab were initiated, and GVHD prophylaxis was changed from sirolimus to tacrolimus. Due to very aggressive malignancy at diagnosis, steroids were weaned aggressively in these three patients as tolerated (courses were 65, 56, and 61 days respectively). GVHD prophylaxis was also stopped prior to Day +100 in all. Perhaps due to this aggressive weaning schedule, in these 3 patients, 1 patient developed recurrent acute GVHD and the other two developed chronic GVHD. The fourth patient was plagued by prolonged anorexia, and was never diagnosed with GVHD nor treated with prolonged systemic therapy for GVHD and yet recovered completely, however the consensus of extramural reviewers was that she had grade III acute GVHD. Another patient was diagnosed by the care team and GI consult as CMV enteritis, but external auditors suggested that isolated GI GvHD as a possible etiology, so the coding was changed to acute GvHD. The final patient was diagnosed as toxic epidermal necrolysis (TEN), but consensus of external reviewers felt that acute GvHD was the more likely etiology, therefore the coding was changed to acute GvHD. Of these 7 patients coded as grade II-IV acute GVHD, one died of ARDS and another of progressive disease, the rest remain alive as of December 2015. Although the rate of moderate to severe acute GVHD was similar to the rate on the previous study HIFLEX, maraviroc was added in Amendment 3.0 in an attempt to reduce the incidence of significant gut and liver inflammation without further inhibiting T-cell activation and proliferation, which had the potential to reduce acute GVHD. However, 4 of 5 patients who received maraviroc developed acute GVHD grade III or IV; this is compared to only 7 patients developing GVHD grade III or IV in the 36 patients transplanted prior to the addition of maraviroc. Therefore, maraviroc use was discontinued. Prior to amendment 5.0, as of date April 4, 2018, preliminary analysis showed a significantly higher rate of GVHD grade III-IV in patients who received maraviroc (4 of 5 = 80%) compared to those patients who did not receive maraviroc (12 of 53 = 25%)(p = 0.02).  
Prior to amendment 2.0, 8 of 26 patients (31%) have developed chronic GVHD (5 mild, 3 moderate by NIH criteria). Six of the 8 patients had oral involvement, with all other sites less commonly involved. Three have required systemic immunosuppressive therapy.
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3.0 protocol eligibility criteria 3.1

3.1.1 Age less than or equal to 21 years. 3.1.2 Does not have a suitable HLA-matched sibling donor (MSD) or volunteer HLAmatched unrelated donor (MUD) available in the necessary time for stem cell donation, or is not a candidate for MSD or MUD HCT due to refractory disease.  
3.1.3 Has a suitable single haplotype matched (≥ 3 of 6) family member donor.  
3.1.4 High risk hematologic malignancy.  
3.1.4.1 High risk ALL in CR1.
Examples include, but not limited to: t(9;22), hypodiploid, MRD >1% at the end of induction, M2 or greater marrow at the end of induction, Infants with MLL fusion or t(4;11).
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3.1.4.2 all in high risk

Examples include, but not limited to t(9;22), BM relapse <36 mo CR1, TALL,
very early (< 6mo CR1) isolated CNS relapse.  
3.1.4.3 ALL in CR3 or subsequent. 3.1.4.4 AML in high risk CR1 (diagnosis of AML includes myeloid sarcoma).  
Examples include but not limited to: preceding MDS, 5q-, -5, -7, FAB M6, FAB M7 not t(1;22), MRD > or = 5% on day 22 (AML08), MRD >
0.1% after two cycles of induction, M3 marrow after once cycle of induction, M2 marrow after two cycles of induction, FLT3-ITD.  
3.1.4.5 AML in CR2 or subsequent.
3.1.4.6 AML in relapse with <25% blasts in BM 3.1.4.7 Therapy related AML, with prior malignancy in CR > 12mo 3.1.4.8 MDS, primary or secondary 3.1.4.9 NK cell, biphenotypic, or undifferentiated leukemia in CR1 or subsequent.  
3.1.4.10 CML in accelerated phase, or in chronic phase with persistent molecular positivity or intolerance to tyrosine kinase inhibitor.  
3.1.4.11 Hodgkin lymphoma in CR2 or subsequent after failure of prior autologous HCT, or unable to mobilize stem cells for autologous HCT.  
3.1.4.12 Non-Hodgkin lymphoma in CR2 or subsequent after failure of prior autologous HCT, or unable to mobilize stem cells for autologous HCT.  
3.1.4.13 JMML 3.1.4.14 One of the following hematologic malignancies that are refractory
(includes chemoresistant relapse or primary induction failure).  
- ALL - AML - CML (blast crisis) - Hodgkin or non-Hodgkin lymphoma 3.1.5 If prior CNS leukemia, it must be treated and in CNS CR  
3.1.6 Does not have any other active malignancy other than the one for which this HCT is indicated.  
3.1.7 No prior allogeneic HCT, and no autologous HCT within the previous 12 months.
3.1.8 Patient must fulfill pre-transplant evaluation:
3.1.8.1 Left ventricular ejection fraction > 40%, or shortening fraction ≥ 25%.  
3.1.8.2 Creatinine clearance (CrCl) or glomerular filtration rate (GFR) ≥ 50 ml/min/1.73m2.
3.1.8.3 Forced vital capacity (FVC) ≥ 50% of predicted value; or pulse oximetry
≥ 92% on room air if patient is unable to perform pulmonary function testing.  
3.1.8.4 Karnofsky or Lansky (age-dependent) performance score ≥ 50 (See APPENDIX A).  
3.1.8.5 Bilirubin ≤ 3 times the upper limit of normal for age. 3.1.8.6 Alanine aminotransferase (ALT) ≤ 5 times the upper limit of normal for age.  
3.1.8.7 Not pregnant. If female with child bearing potential, must be confirmed by negative serum or urine pregnancy test within 14 days prior to enrollment.  
3.1.8.8 Not breast feeding 3.1.8.9 Does not have current uncontrolled bacterial, fungal, or viral infection.
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3.2 inclusion criteria for haploidentical

3.2.1 At least single haplotype matched (≥ 3 of 6) family member  
3.2.2 At least 18 years of age.  
3.2.3 HIV negative.  
3.2.4 Not pregnant as confirmed by negative serum or urine pregnancy test within 14 days prior to enrollment (if female).  
3.2.5 Not breast feeding. 3.2.6 Regarding eligibility, is identified as either:
3.2.6.1 Completed the process of donor eligibility determination as outlined in 21 CFR 1271 and agency guidance; OR
3.2.6.2 Does not meet 21 CFR 1271 eligibility requirements, but has a declaration of urgent medical need completed by the principal investigator or physician sub-investigator per 21 CFR 1271.
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3.3 gender and minorities

According to institutional and NIH policy, this study will accession patients regardless of gender and ethnic background. Institutional experience confirms broad representation in this regard. However, pregnant and lactating females are excluded from participation as the short and long-term effects of the preparative agents, study infusion (recipients) as well as the long-term effects of mobilization and apheresis procedure (donors) on a fetus and a nursing child through breast milk are not entirely known at this time.
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4.0 treatment plan

| 4.1                                                                                                                                | Preparative regimen  DAY MEDICATION   | DOSE                                                                | DOSE #   |
|------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------|---------------------------------------------------------------------|----------|
| -9                                                                                                                                 | Total Lymphoid Irradiation            | 2Gy per fraction x 2 fractions                                      | 1,2 of 4 |
| (TLI)  Total Lymphoid Irradiation                                                                                                  | 2Gy per fraction x 1 fraction         | 3 of 4                                                              |          |
| -8                                                                                                                                 | (TLI)  Fludarabine                    | 30 mg/m2 intravenous once daily                                     | 1 of 5   |
| Total Lymphoid Irradiation                                                                                                         | 2Gy per fraction x 1 fraction         | 4 of 4                                                              |          |
| -7                                                                                                                                 | (TLI)  Fludarabine                    | 30 mg/m2 intravenous once daily                                     | 2 of 5   |
| -6                                                                                                                                 | Cyclophosphamide                      | 60 mg/kg intravenous once daily                                     | 1 of 1   |
| Fludarabine                                                                                                                        | 30 mg/m2 intravenous once daily       | 3 of 5                                                              |          |
| -5                                                                                                                                 | Fludarabine                           | 30 mg/m2 intravenous once daily                                     | 4 of 5   |
| -4                                                                                                                                 | Fludarabine                           | 30 mg/m2 intravenous once daily                                     | 5 of 5   |
| -3                                                                                                                                 | Thiotepa                              | 5 mg/kg intravenous twice daily                                     | 1,2 of 2 |
| -2                                                                                                                                 | Melphalan                             | 70 mg/m2 intravenous once daily                                     | 1 of 2   |
| -1                                                                                                                                 | Melphalan                             | 70 mg/m2 intravenous once daily                                     | 2 of 2   |
| 0                                                                                                                                  | HPC,A Infusion(CD34+  selected)       |                                                                     |          |
| +1                                                                                                                                 | HPC,A infusion (CD45RA  depleted)     |                                                                     |          |
| +2-5                                                                                                                               | --                                    |                                                                     |          |
| +6                                                                                                                                 | TC-NK Infusion                        | 5mcg/kg subcutaneous or intravenous daily until                     |          |
| +7                                                                                                                                 | G-CSF                                 | ANC >2000 for 2 consecutive days, and then as  clinically indicated |          |
| +13                                                                                                                                | Mycophenylate mofetil                 | 15 mg/kg intravenously three times daily.                           |          |
| Cellular infusion procedures and monitoring:  For the proper infusion procedures and monitoring of the HPC product please refer to |                                       |                                                                     |          |  
For the proper infusion procedures and monitoring of the HPC product please refer to BMT&CT SOP 40.02 "Hematopoietic Progenitor Cell Infusion - FRESH (Allogeneic): IV
Push and IV Drip" or SOP 40.03 "Hematopoietic Progenitor Cell Infusion - FROZEN: IV
Push". For the TC-NK infusion, please see BMT&CT SOP 40.04 "Therapeutic Cell Infusion". Importantly, for TC-NK infusion, steroid pre-medications should not be given unless specified by an attending transplant physician. The TC-NK product will be infused by slow IV push over 3 to 15 minutes. Please note that all relevant SOPs can be found on the BMT&CT Clinical Transplant Program intranet page: http://home.web.stjude.org/bone_marrow/clinicalHome.shtml Importantly, during the cellular infusions, monitoring of vital signs, breath sounds, heart rate, pulse oximetry, and I/O will be done per the established transplant nursing procedure, as well as appropriate Department of BMT&CT SOPs, then documented on the Cellular Product Infusion Record. If a reaction is suspected at any time during the infusion, the nurse will 1. Stop the infusion, 2. Notify the Attending Transplant Physician immediately, 3. NOT discard the product until physician orders are given. Proper documentation (symptoms of patient, vital signs, actions taken, outcome, and follow-up) will be completed in the Cellular Product Infusion Record.
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general comments

The Hematopoietic Progenitor Cell, Apheresis (HPC,A) infusion may be delayed by approximately 24 hours in order to accommodate progenitor cell collection with the donor, the Blood Donor Center and/or HAL as well as the research participant clinical condition.  
The administration of the TC-NK infusion may also be adjusted in order to accommodate NK-cell collection with the donor, the Blood Donor Center and/or HAL as well as the research participant clinical condition. Depending on date of NK-cell collection, it will be acceptable for the TC-NK infusion to occur between and including days +5 and +9 post progenitor cell infusion. In the event that the date of NK-cell infusion is adjusted, the mycophenylate mofetil (MMF) would typically be similarly adjusted such that dosing is initiated seven days post NK-cell infusion.  
The term "every" used in tables is an approximate term meaning that these medications noted will be administered approximately "every" 12 hours. The drug administration timing in the case of "every 12 hours" may be modified by approximately +/- 4 hours or as clinically indicated such as in the case of surgical procedures or to accommodate other necessary medication, blood product delivery, or procedures (such as a needed CT scan). The term "day" does not refer to an absolute calendar day. It refers to a general 24-hour period. Dosing for the medications cyclophosphamide, fludarabine, thiotepa, and melphalan may be modified for research recipients based upon actual body weight or adjusted ideal body weight when clinically indicated. Mesna will be administered for prevention of hemorrhagic cystitis from the medication cyclophosphamide. In general, mesna is administered at 15 mg/kg/dose prior to cyclophosphamide and at approximately 3, 6, and 9 hours after the cyclophosphamide infusion, to give a 1:1 ratio of mesna:cyclophosphamide. Mesna dose and administration schedule may vary based on physician recommendation.  
Criteria for medication calculations based on body weight/body surface area and other medication related information can be found in the St. Jude Formulary http://www.crlonline.com/crlsql/servlet/crlonline or the St. Jude Dept of Pharmaceutical Sciences intranet website http://home.web.stjude.org/pharmaceutical_ser/drugInfo.shtml. Medication doses may be rounded to the nearest integer or to the nearest appropriate quantity when clinically or pharmaceutically indicated as per the MD and PharmD.  
TLI will be given at 800cGy total dose in 4 fractions over 2 or 3 days. Fractions are given at a minimum of 6 hours apart from beam on to beam on. In order to facilitate scheduling, the general procedure for administering TLI has been defined here and in the above table. It should be noted that TLI can be administered at any point during the preparative regimen prior to the progenitor cell infusion, and over as many days as needed to further accommodate scheduling issues in collaboration with the Division of Radiation Oncology and will not be considered a variance in protocol if the total dose of 800 cGy is met.  
G-CSF will generally start on Day +7, but initial dose may be moved to an earlier or later day as to not interfere with NK cell infusion. G-CSF may also be dose adjusted or stopped early if rapid engraftment is considered to be potentially harmful to the patient (such as with engraftment syndrome) in the opinion of the PI or transplant care provider.  
MMF for GVHD prophylaxis: MMF will start one week after the NK cell infusion. In the event NK cells cannot be given, MMF may start Day 0, or as soon as it is known that the NK cell graft is unavailable (if later than Day 0). MMF is typically given IV initially, but may be converted to oral dosing when the patient is tolerating oral medications. In the absence of GVHD, MMF will be discontinued approximately Day +60. However, this regimen may be modified as clinically indicated in the presence of other factors including but not limited to persistent neutropenia, mixed or decreasing donor chimerism, graft rejection/failure or persistent/recurrent malignancy. In the event the participant develops GVHD, treatment will be according to the SOPs of the St. Jude Department of BMT&CT.
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4.2 donor selection

If more than one family member donor is acceptable, then donor selection will be based on the preference of the primary transplant attending. Factors in selection will include donorrecipient matching of CMV serology, donor-recipient red blood cell compatibility, degree of HLA matching, size of the potential donor, previous use as a donor, presence of donorspecific antibody, and overall health of the potential donor.  
Donor eligibility for cell collection will be determined through the guidelines outlined in 21 CFR 1271 and the Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps). Potential donors will undergo an initial screening process that will include at least a complete physical exam, history and testing for relevant communicable diseases. Physical exams to evaluate donor candidacy will be conducted by a non-Department of BMTCT physician (St. Jude or non-St. Jude). For subsequent therapeutic cell collection procedures, if a complete screening procedure has been performed within the previous 6 months, an abbreviated donor screening procedure may be used for these repeat donations. The abbreviated screening procedure must determine and document any changes in the donor's medical history since the previous donation that would make the donor ineligible, including changes in relevant social behavior.  
If a donor is determined to be ineligible, the donor is not automatically excluded. Part 21 CFR 1271.65 (b)(1)(i) allows use of ineligible donors who are first or second degree blood relatives. In this situation, the physician will document the necessity of using the ineligible donor by providing a statement of "Urgent Medical Need" as explained in the 21 CFR 1271.3 (u). The cell therapy products will be labeled as required in 21 CFR 1271.65 (b)(2). Recipients or their legal guardians will be informed of the use of an ineligible donor.  
Please see Departmental SOP 30.05 "Determination of Eligibility and Suitability for Stem Cell and Therapeutic Cell Allogeneic and Autologous Donors" for additional information.
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4.3 donor mobilization and graft

A G-CSF mobilized peripheral blood stem cell (PBSC) product (identified as HPC,A) is the preferred progenitor cell graft source. Our desired target goal will be 3-5 x 106 CD34+ cells/kg. This number of cells will be necessary to provide an adequate graft, following the various ex vivo manipulations, for prompt reconstitution. Two days of collection are typically needed to achieve this goal. However, on rare occasions, additional days may be necessary. Donors will undergo a standard hematopoietic stem cell mobilization regimen consisting of 6 days of G-CSF given subcutaneously at 10 micrograms/kilogram. The graft will be collected by leukapheresis on days 5 and 6. The HPC product will typically be collected and infused fresh, however there may be patients or logistical situations that require the HPC product to be collected early, processed, and stored frozen. The NK-cell product will always be collected, processed, and infused fresh.  
The decision to use a fresh versus frozen HPC will be made by the PI and/or primary  
transplant attending based on patient and donor factors, as well as potential scheduling conflicts.  
DONOR MOBILIZATION TIME LINE (if fresh HPC product)  
| conflicts.                                                   | DONOR MOBILIZATION TIME LINE (if fresh HPC product)   |                         |
|--------------------------------------------------------------|-------------------------------------------------------|-------------------------|
| DAYS                                                         | MEDICATION                                            | APHERESIS               |
| Day -5                                                       | G-CSF 10 mcg/kg/day SC*                               |                         |
| Day -4                                                       | G-CSF 10 mcg/kg/day SC*                               |                         |
| Day -3                                                       | G-CSF 10 mcg/kg/day SC*                               |                         |
| Day -2                                                       | G-CSF 10 mcg/kg/day SC*                               |                         |
| Day -1                                                       | G-CSF 10 mcg/kg/day SC*                               | Apheresis for HPC graft |
| Day 0                                                        | G-CSF 10 mcg/kg/day SC*                               | Apheresis for HPC graft |
| Day +1-4                                                     | Rest day                                              |                         |
| Day +5                                                       | Apheresis for TC-NK                                   |                         |
| * G-CSF may be reduced if the donor's WBC is >75.0 x 106 /mL |                                                       |                         |  
The dose of G-CSF may require modification based on the complete blood counts
(CBC). If the donor's white blood count (WBC) is >75.0 x 106/ml the dose of cytokine administered will be reduced. The guidelines for dose modification can be found in the St. Jude Children's Research Hospital Department of BMT and CT SOP 30.06.00 "The practice for the evaluation, preparation and care of allogeneic and autologous donors mobilized with growth factor." Ongoing updates of this document can be located at the following St. Jude intranet website: http://home.web.stjude.org/bone_marrow/
The daily leukapheresed volumes for PBSC collection is generally 3–4 total blood volumes based on CD34+ cell counts. Two additional days of leukapheresis may be performed at the physician's discretion (no more than 4 total) to reach the cell dose target, however, this is expected to be rare.  
The TC-NK will be collected by the same leukapheresis procedures as the HPC product.  
However the donor will not undergo a mobilization procedure for this product collection. NK cell product collection will be limited to one day. Collection will typically occur on day +5, but may occur anywhere from day +4 to day+8 depending on patient and donor factors.  
Leukapheresis may be terminated early upon request of donor, or when deemed medically necessary per the judgment of the treating sub-investigator physician or principal investigator. All PBSC products will be collected as per Foundation for the Accreditation of Cellular Therapy (FACT) guidelines. Donors will be monitored during the period of the mobilization and leukapheresis procedure with appropriate laboratory evaluation (Appendix D).  
If we are unable to collect the minimum dose of 2 x 106 CD34+ cells/kg of recipient weight from the first donor, and the recipient has not yet initiated the preparative regimen, then an alternative family member may be used if he/she fulfills all the donor criteria described in section 3.2. If the donor is unwilling or unable to complete the mobilization process or leukapheresis procedure, a BM product may be used. The BM product will be processed using the same cell selection methodology on the CliniMACS device.
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4.4 graft preparation

Graft evaluation and preparation will take place in the Human Applications Laboratory
(HAL) in the Department of Therapeutics Production and Quality (TPQ) using established SOP.  
The initial HPC product(s) will be TCD using the investigational CliniMACS device and CD34 Microbead reagent as directed by the manufacturer (Miltenyi Biotech). See section 5.2 for additional CliniMACS device information. Briefly, hematopoietic progenitor cells collected by apheresis (HPC,A) from the mobilized donor will be initially assessed in the HAL and stored overnight at 4oC. The next morning, the product will be washed to remove platelets and adjusted to an appropriate cell concentration for incubation with the CliniMACS CD34 Microbead reagent in the manufacturer provided media. The cells will be washed to remove unbound microbeads. These cells will be applied to the CliniMACS device and the enrichment will be performed using the program "CD34 Enrichment 2.1" as described by the manufacturer.  
After enrichment is complete, the cells will be washed and resuspended in an infusion grade solution. The graft product will be enumerated and assessed for viable CD34+ cell and CD3+ T-cell content by flow cytometry. The processed HPC,A product will be infused fresh or frozen for future use after completion of release testing and evaluation. Cryopreservation will be performed per SOPs of the Human Applications Laboratory. Target cell doses are listed in the following table:
HPC Graft Target Dose Minimum Dose Maximum Dose CD34+ cells/kg ≥ 2 x 106 2 x 106 50 x 106 CD3+ cells/kg ≤ 0.05 x 106 0.001 x 106 0.1 x 106 If the target dose is not achieved in a single collection, up to two additional collections will be performed for CD34+ enrichment (see Section 4.3). Once the target dose is obtained for the CD34+ enriched product, one additional day of apheresis will be performed. This HPC product will be processed for CD45RA+ depletion using the investigational CliniMACS device as directed by the manufacturer (Miltenyi Biotech). See section 5.2 for additional CliniMACS device information. Briefly, therapeutic cells collected by apheresis (HPC,A) from the mobilized donor will be initially assessed in the HAL and stored overnight at 4oC. The next morning, the product will be washed to remove platelets and adjusted to an appropriate cell concentration for incubation with the CliniMACS CD45RA Microbead reagent in the manufacturer provided media. The cells will be washed to remove unbound microbeads. These cells will be applied to the CliniMACS device and the depletion will be performed using the "Depletion 3.1" software as described by the manufacturer. There is no target CD34+ dose for the CD45RA depleted product. Given that the previously infused CD34+ enriched product(s) will provide a minimum dose of 2 x 106/kg, once the CD45RA depleted product is infused, the patient is likely to have received 3-5 x 106 CD34+ cells/kg.  
There is no CD3+ cell target dosing on the CD45RA+ depleted product, the goal of this depletion is a ≥ 2 log10 depletion of CD45RA+ cells, however a depletion that does not achieve this target level may still be released to the patient as long as the maximum CD3+CD45RA+ dose is not exceeded. The maximum dose of CD3+CD45RA+ cells will be limited to no greater than 0.05x106/kg.  
HPC Graft Target Dose Minimum Dose Maximum Dose CD3+CD45RA+ cells/kg ≤0.05 x 106 N/A 0.05x106 In rare instances, the progenitor cell dose from the first day collection may be so high that it is feasible to split that product in two aliquots such that one aliquot may undergo CD34 enrichment, and the second aliquot may undergo CD45RA-depletion, thereby deriving both progenitor cell grafts from a single collection. This is allowed at the discretion of the PI, and both products may be infused on the same day when derived from a single collection.  
| HPC Graft                                                                                | Target Dose                                       | Minimum Dose       | Maximum Dose        |
|------------------------------------------------------------------------------------------|---------------------------------------------------|--------------------|---------------------|
| CD34+ cells/kg                                                                           | 3-5 x 106 (combined)                              | 2 x 106 (combined) | 50 x 106 (combined) |
| CD45RA+                                                                                  | ≥ 2 log10 depletion (CD45RA+ depleted graft only) |                    |                     |
| CD3+CD45RA+ cells/kg                                                                     | ≤0.05 x 106                                       | N/A                | 0.05x106            |
| In rare instances, the progenitor cell dose from the first day collection may be so high |                                                   |                    |                     |  
The therapeutic NK-cell product (TC-NK) will be processed using the investigational CliniMACS device as directed by the manufacturer (Miltenyi Biotech). See section 5.2 for additional CliniMACS device information. Briefly, therapeutic cells collected by apheresis (TC,A) from the non-mobilized donor will be initially assessed in the HAL and stored overnight at 4oC. The next morning, the product will be washed to remove platelets and adjusted to an appropriate cell concentration for incubation with the CliniMACS CD3 Microbead reagent in the manufacturer provided media. The cells will be washed to remove unbound microbeads. These cells will be applied to the CliniMACS device and the depletion will be performed using the "Depletion 2.1" software as described by the manufacturer. In the second purification step, the CD3+ depleted product will be enriched for CD56+ cells using the CliniMACS immunomagnetic enrichment program 1.1 with the CliniMACS CD56 Microbead reagent.  
For this protocol, our goal will be to infuse immediately after processing (Day +6) all the NK cells collected to give >2x106 CD56+ cells/kg of recipient body weight, but allowing for a CD3+ CD56- cell dose of no greater than 0.05x106/kg. The target doses for the NKcell product are as follows:
TC-NK Target Dose Minimum Dose Maximum Dose  
| TC-NK          | Target Dose                                     | Minimum Dose   | Maximum Dose   |
|----------------|-------------------------------------------------|----------------|----------------|
| CD56+ cells/kg | > 2 x 106                                       | 0.1 x 106      | 400 x 106      |
| CD3+ cells/kg  | ≤0.05 x 106                                     | N/A            | 0.05 x 106     |
| 4.5            | Additional Progenitor Cell Graft Administration |                |                |  
Infusion of an additional HPC graft from the original or an alternative haploidentical donor may be performed for participants when clinically indicated for graft failure, poor immune reconstitution, or poor hematopoietic recovery. The use of and content of a conditioning regimen is left to the discretion of the PI and/or primary transplant attending such that the most appropriate therapy is chosen for the clinical situation.  
The HPC graft will typically be obtained by apheresis (HPC,A) and be infused fresh.  
The target dose for this additional CD 34+ infusion is ≥5 x 106 cells/kg. If the participant has quiescent or active BOOP, acute Grade III-IV GVHD, or any other reason that a severely T-cell depleted graft may be indicated, then a graft from the donor processed on the CliniMACSTM device using either CD34+ selection (using established SOPs) or CD3+ depletion methodology may be utilized. The boost target dose for these patients is
≥10 x 106 CD34+cells/kg with a CD3+ cell/kg dose of ≤ 0.5 x 105 CD3+ cells/kg.
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4.6 donor lymphocyte infusions

Donor lymphocyte infusions (DLI) may be given with or without preceding lymphodepleting chemotherapy. Recipients who are further out from transplant and with substantial lymphocyte engraftment may quickly tolerize the newly infused donor lymphocytes. In such patients, lymphodepleting chemotherapy may be needed for effective DLI activity. Regimen will vary based on patient condition, underlying disease, etc. Regimen will be determined by attending physician and/or PI, but in general would be low dose, expected to have minimal direct toxicity, and have limited or very transient effects on ANC. Regimens for lymphodepleting chemotherapy will be defined in a separate document (non-protocol treatment plan-NPTP) and will require additional consent to the NPTP.  
4.6.1 Conventional Donor Lymphocyte Infusions DLI may be administered from the original donor for decreased donor chimerism, serious viral reactivation or infection, or any evidence of disease.  
- Decreased donor chimerism is defined as any single chimerism test that is not 100% donor
- Serious viral reactivation is defined as any virus detected in the blood by PCR, with a 1 log increase in viral load despite antiviral medication.  
- Serious viral infection is defined as any infectious disease from an identified virus that has progressed in severity despite antiviral therapy (if available) or shows no improvement despite 1 week of antiviral therapy (if available)
- Any evidence of disease is defined as any flow cytometry, PCR, NGS
(such as RNAseq), cytogenetic, or any other validated molecular testing that detects the presence of the original hematologic malignancy within the limits of that particular test.  
The DLI may be collected as a whole blood unit donation or by leukapheresis. If the DLI is collected by standard phlebotomy, the volume to be collected would be approximately 300 ml whole blood. If the DLI is collected by leukapheresis, the amount to be processed would be approximately 2 total blood volumes.  
Prior to administration of DLI, the immunosuppression should be withdrawn and the recipient should have no active GVHD. The initial dose will typically be 2.5 x 104 CD3+/kg. Subsequent doses will be administered at approximately 2 to 4week intervals with escalating doses of T cells if no moderate or severe GVHD occurs with the prior DLIs. The typical initial dose escalation for patients on this protocol is presented in the following table:
CONVENTIONAL DLI DOSE AND SCHEDULE
DLI Dose(104 **CD3+/kg) Comments**
Initial Dose 2.5 Approximately 2-4 week interval  
| CONVENTIONAL DLI DOSE AND SCHEDULE                                                  |                   |                                 |
|-------------------------------------------------------------------------------------|-------------------|---------------------------------|
| DLI                                                                                 | Dose(104 CD3+/kg) | Comments                        |
| Initial Dose                                                                        | 2.5               | Approximately 2-4 week interval |
| Dose #2                                                                             | 5                 | If no moderate or severe GVHD   |
| Dose #3                                                                             | 10                |                                 |
| Although this algorithm will be appropriate for a majority, the treating transplant |                   |                                 |  
Dose \#3 10 Although this algorithm will be appropriate for a majority, the treating transplant attending physician may alter the dose and/or interval of DLI based on response to previous DLI, the severity of the clinical situation, and the condition of the patient. There is a low threshold for providing an initial dose of 2.5 x 104 CD3+/kg. DLI at this dose level have not been associated with GVHD.  
Therefore, DLI may be initiated for any chimerism less than 100% in a patient who is off immune suppression. The risk of GvHD increases with increasing doses. In the last 5 years, no patient has developed moderate or severe GVHD
after haploidentical DLI dosed at 5 x 104 CD3+/kg, though mild GVHD has occasionally occurred. Doses of 10 x 104 CD3+/kg should be considered to carry a risk of severe GVHD. Therefore dose escalation to this level must incorporate the appropriate risk/benefit analysis by the treating transplant physician.  
4.6.2 CD45RA-depleted donor lymphocyte infusions Low dose conventional DLI has been found to be very effective for treatment of low level mixed chimerism, but it's efficacy in serious viral infections and recurrent disease (even at MRD levels) is poor. Although a median of 108 memory T-cells are adoptively transferred at the time of transplant on this study, and rapid donor immune reconstitution has been previously demonstrated, some patients may have recurrence or persistence of lymphopenia due to infections or posttransplant immunosuppressive drugs (such as corticosteroids). Therefore selected patients may benefit from additional adoptive transfer of donor memory cell populations in the post-transplant setting. This would include: 1. Patients with viral infections that are not responding to antiviral therapy or historically have a poor response to antiviral therapy; 2. Patients who have evidence of relapse, particularly molecular or MRD levels of relapse; 3. Patients who are severely lymphopenic, such as ALC <500/uL and/or T-cell count <300/uL. These patients will be allowed to receive CD45RA-depleted DLI in the following doses:  
| CD45RA-DEPLETED DLI DOSE AND SCHEDULE                                           |                   |                                 |
|---------------------------------------------------------------------------------|-------------------|---------------------------------|
| DLI                                                                             | Dose(106 CD3+/kg) | Comments                        |
| Initial Dose                                                                    | 0.1               | Approximately 2-4 week interval |
| Dose #2                                                                         | 1                 | If no moderate or severe GVHD   |
| Dose #3                                                                         | 10                |                                 |
| The DLI may be collected as a whole blood unit donation or by leukapheresis. If |                   |                                 |  
Dose \#3 10 The DLI may be collected as a whole blood unit donation or by leukapheresis. If the DLI is collected by standard phlebotomy, the volume to be collected would be approximately 300 ml whole blood. If the DLI is collected by leukapheresis, the amount to be processed would be approximately 2 total blood volumes. The collected donor lymphocytes would then undergo CD45RA depletion as decribed for the HPC,A graft (section 4.4).  
The release criteria of the CD45RA-depleted DLI will include at least a ≥ 2 log10 depletion of CD45RA+ cells. The maximum dose of CD3+CD45RA+ cells will be limited to no greater than 0.05x106/kg for any CD45RA-depleted DLI.  
Conventional DLI and CD45RA-depleted DLI cannot be given within 14 days of each other.
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4.7 quality assurance of cellular

Quality assurance for cell products is overseen by the TPQ Quality Assurance division, which authorizes release of all products. Only trained stem cell processors will process the cell products. A labeling and product tracking system is in place to ensure that the correct cells are infused into the research participant.  
Assays of cell numbers and immunophenotyping will be performed both before cell processing and at critical stages of the process. These values will be recorded according to SOP of the HAL. All products will be tested for viability and sterility (culture and Gram stain). Culture results are not available before infusion of cell products. If the gram stain is positive, the research participant/parent and/or guardian will be informed of this event and of the risks of proceeding prior to infusion. Positive results will be investigated as per the variance procedures of the HAL. The IRB and FDA will be notified, if at any time after infusion, cell product was determined to be contaminated.
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5.0 medication and device information

| 5.1           | Medications  Cyclophosphamide (Cytoxan) Source &  Cyclophosphamide is a nitrogen mustard derivative.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | It acts as an   |
|---------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|
| Pharmacology  | alkylating agent that causes cross-linking of DNA strands by binding with  nucleic acids and other intracellular structures, thus interfering with the  normal function of DNA. It is cell cycle, phase non-specific.  Cyclophosphamide is well absorbed from the GI tract with a  bioavailability of >75%. It is a prodrug that requires activation. It is  metabolized by mixed function oxidases in the liver to 4- hydroxycyclophosphamide, which is in equilibrium with aldophosfamide.  Aldofosfamide spontaneously splits into nitrogen mustard, which is  considered to be the major active metabolite, and acrolein. In addition,  4hydroxycy-clophosphamide may be enzymatically metabolized to  4ketocyclophosphamide and aldophosfamide may be enzymatically  metabolized to carboxyphosphamide that is generally considered inactive.  Cyclophosphamide and its metabolites are excreted mainly in the urine.  Dose adjustments should be made in patients with a creatinine clearance  of <50 ml/min. |                 |
| Formulation   | Cyclophosphamide is available in vials containing 100, 200, 500, 1000                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                 |
| and Stability | and 2000mg of lyophilized drug and 75 mg mannitol per 100 mg of  cyclophosphamide. Both forms of the drug can be stored at room  temperature. The vials are reconstituted with 5, 10, 25, 50 or 100 ml of  sterile water for injection, respectively, to yield a final concentration of 20  mg/ml. Reconstituted solutions may be further diluted in either 5%  dextrose or 0.9% NaCl containing solutions. Diluted solutions are  physically stable for 24 hours at room temperature and 6 days if  refrigerated, but contain no preservative, so it is recommended that they  be used within 24 hours of preparation.                                                                                                                                                                                                                                                                                                                                                                                             |                 |  
| Supplier                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | Commercially available                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Toxicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Dose limiting toxicities of cyclophosphamide includes BM suppression  and cardiac toxicity. Cardiac toxicity is typically manifested as  congestive heart failure, cardiac necrosis or hemorrhagic myocarditis and  can be fatal. Hemorrhagic cystitis may occur and necessitates  withholding therapy. The incidence of hemorrhagic cystitis is related to  cyclophosphamide dose and duration of therapy. Forced fluid intake  and/or the administration of mesna decreases the incidence and severity of  hemorrhagic cystitis. Other toxicities reported commonly include nausea  and vomiting (may be mild to severe depending on dosage), diarrhea,  anorexia, alopecia, immunosuppression and sterility. Pulmonary fibrosis,  SIADH, anaphylaxis and secondary neoplasms have been reported rarely. |
| Route                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Intravenous infusion                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| Fludarabine (Fludara)  Source &  Fludarabine phosphate is a synthetic purine nucleoside analog. It acts by  Pharmacology  inhibiting DNA polymerase, ribonucleotide reductase and DNA primase  by competing with the physiologic substrate, deoxyadenosine  triphosphate, resulting in inhibition of DNA synthesis. In addition,  fludarabine can be incorporated into growing DNA chains as a false base,  thus interfering with chain elongation and halting DNA synthesis.  Fludarabine is rapidly dephosphorylated in the blood and transported  intracellularly via a carrier-mediated process. It is then phosphorylated  intracellularly by deoxycytidine kinase to the active triphosphate form.  Approximately 23% of the dose is excreted as the active metabolite in the  urine (with dosages of 18-25 mg/m2 /day for 5 days). Renal clearance  appears to become more important at higher doses, with approximately  41-60% of the dose being excreted as the active metabolite in the urine  with dosages of 80-260 mg/m2 .  Formulation  Fludarabine is supplied in single-dose vials containing 50 mg fludarabine  and Stability  as a white lyophilized powder and 50 mg of mannitol. The intact vials  should be stored under refrigeration. Each vial can be reconstituted by  adding 2 ml of sterile water for injection resulting in a final concentration  of 25 mg/ml. Because the reconstituted solution contains no antimicrobial  preservative, the manufacturer recommends that it should be used within 8  hours of preparation. The solution should be further diluted in 5%  dextrose or 0.9% NaCl prior to administration.  Supplier Commercially available. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |  
| T o xicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | T he                                      | maj or d ose   | li miti n g t o xicit y of fl u dara bi ne is   | m yel os u p pressi o n.   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------|----------------|-------------------------------------------------|----------------------------|
| Na usea a n d v o miti n g are us uall y mil d.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Si de eff ects re p orte d c o m m o nl y |                |                                                 |                            |
| i ncl u de a n ore xia, fe v er a n d c hills, al o pecia a n d r as h.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Ne ur ot o xicit y ca n be                |                |                                                 |                            |
| ma nifeste d b y s o m n ole n ce, f ati g ue, p eri p her al n e ur o pat h y, me nt al stat us  c ha n ges, c ortical bli n d ness a n d c o ma a n d is m ore c o m m o n at hi g h d oses.  Ne ur ot o xicit y is us uall y dela ye d, occ urri n g 2 1 6 0 da ys aft er t h e  c om pleti o n of a c o urse of t hera p y a n d m a y be irre versi ble. Si de eff ects  re p orte d less c o m m o nl y i ncl u de diarr hea, st o matitis, i ncrease d li ver  f u ncti o n tests, li ver fail ure, c hest pai n, arr h yt h mias a n d seiz ures.  P ul m o nar y t o xicit y i ncl u des aller gic p ne u m o nitis c haracteri ze d b y c o u g h,  d ys p nea, h y p o xia a n d p ul m o nar y i nfiltrates. Dr u g i n d uce d p ne u m o nitis is  a dela ye d eff ect, occ urri n g 3 2 8 da ys after t he a d mi nistrati o n of t he t hir d  or later c o urse of t her a p y. A d mi nistrati o n of c ortic oster oi ds us u all y  res ults in res ol uti o n of t hese s y m pt o ms. |                                           |                |                                                 |                            |
| R o ute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | I ntr a ve n o us                         |                |                                                 |                            |  
res ults in res ol uti o n of t hese s y m pt o ms.  
R o ute I ntr a ve n o us  
|                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | G- C S F ( Fil gr asti m, Ne u p o ge n                                             | )         |          |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|-----------|----------|
| S o urce &  P har mac ol o g y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | C S F ( gra n ul oc ytic c ol o n y sti m ulati n g fact or), is a bi os y nt hetic |           |          |
| he mat o p oietic a ge nt t hat is ma de usi n g rec o m bi na nt D N A tec h n ol o g y i n  c ult ures of Esc h eric hi a c oli. G- C S F sti m ulates pr o d ucti o n, mat urati o n  a n d acti vati o n of ne utr o p hils. I n a d diti o n, e n d o ge n o us G C S F e n ha n ces  certai n f u ncti o ns of mat ure ne utr o p hils, i ncl u di n g p ha g oc yt osis,  c he m ota xis a n d a nti b o d y --de pe n d e nt cell ular c yt ot o xicit y.                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                                                                     |           |          |
| F or m ulati o n  a n d Sta bilit y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | C S F is s u p plie d i n vials c o ntai ni n g 3 0 0 mc g a n d 4 8 0              | mc g of G | C S F at |
| a c o nce ntrati o n of 3 0 0 mc g/ ml. T he i ntact vials s h o ul d be st ore d u n d er  refri gerati o n. T he vi als ca n b e left o ut of refri gerati o n f or 2 4 h o urs, b ut  s h o ul d be discar de d if left at r o o m te m perat ure f or l o n ger peri o ds of ti me.  G- C S F ca n be dr a w n u p i nt o t u berc uli n s yri n ges f or a d mi nistrati o n a n d  st ore d u n der refri ger ati o n f or u p t o 7 da ys pri or t o usa ge. G C S F ca n be  f urt her dil ute d f or i ntra v e n o us i nf usi o n i n 5 % de xtr ose. D o n ot dil ute i n  sali ne---pr eci pitate m a y f or m. If t h e fi nal c o nce ntr ati o n of t his pr o d uct is  < 1 5 mc g/ ml, it is rec o m me n de d t hat al b u mi n be a d de d t o a fi nal  c o nce ntr ati o n of 2 m g/ ml ( 0. 2 %) t o mi ni mize a ds or pti o n of t he dr u g t o  i nf usi o n c o ntai ners a n d e q ui p me nt. |                                                                                     |           |          |
| S u p plier                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | C o m merciall y a vaila ble.                                                       |           |          |  
| Toxicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | G-CSF                        | causes   | marked   | leukocytosis.   | Adverse   | reactions   | reported   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|----------|----------|-----------------|-----------|-------------|------------|
| commonly include bone pain, thrombocytopenia, diarrhea, nausea, rash,  alopecia, fever, anorexia and pain or bruising at the injection site.  Allergic reactions, MI, atrial fibrillation, and splenomegaly have been  reported rarely. G-CSF is contraindicated in research participants with  allergy to E. coli derived products.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                              |          |          |                 |           |             |            |
| Route                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Intravenous or subcutaneous. |          |          |                 |           |             |            |
| Melphalan (L-phenylalanine mustard, phenylalanine mustard, L-PAM, L-sarcolysin,  Alkeranâ )  Source &  Melphalan, a derivative of nitrogen mustard, is a bifunctional alkylating  Pharmacology  agent. Its chemical name is 4-[bis(2-chloroethyl)amino]-L-phenylalanine,  and it has a molecular weight of 305.20. Melphalan is active against  tumor cells that are actively dividing or at rest. Its cytotoxicity is thought  to be due to inter-strand cross-linking with DNA, probably by binding at  the N7 position of guanine. Melphalan is highly protein bound and does  not penetrate well into the cerebral spinal fluid. Elimination half-life after  intravenous administration in adults is approximately 75 minutes.  Elimination appears to be primarily by chemical hydrolysis, but caution  should be used in patients with renal impairment. Plasma concentrations  of melphalan after oral administration are highly variable, possibly due to  incomplete absorption, variable "first pass" hepatic metabolism or rapid  hydrolysis. Area under the plasma concentration-time curves for orally  administered melphalan is approximately 60% of intravenously  administered melphalan in adult studies.  Formulation  Available as 2 mg tablets for oral administration. This medication is stable  and Stability  at room temperature until expiration date on the packaging. Intravenous  formulation is supplied as 50 mg freeze dried glass vial. Each 50 mg vial  is supplied in a carton containing a 10 ml vial of sterile diluent.  Lyophilized melphalan should be stored at controlled room temperature  and protected from light. Each vial is marked with its expiration date. The  melphalan for injection must be reconstituted immediately prior to  infusion by rapidly adding the contents of the diluent vial (10 ml) to the  freeze dried powder with a 20 gauge or larger sterile needle and  immediately shaking vigorously until a clear solution is obtained. This  results in a 5 mg/ml solution. The dose should then be diluted in 0.9%  NaCl for injection to a final concentration of not greater than 0.45 mg/ml.  The resulting admixture should be infused over a minimum of 15 minutes.  The infusion should be completed within 60 minutes of reconstitution. Do  not refrigerate the reconstituted melphalan.  Supplier Commercially available |                              |          |          |                 |           |             |            |  
| Toxicities   | Melphalan is cytotoxic and caution should be used in handling and  preparing the solution or administering the tablets. Use of gloves is  recommended, and if contact with skin or mucosa occurs, immediately  wash thoroughly. Second cancers such as acute non-lymphocytic  leukemia, myeloproliferative syndrome, and carcinoma have been  reported in patients taking melphalan alone or in combination with other  chemotherapy or radiation. Melphalan causes suppression of ovarian  function in premenopausal women, with a significant number of patients  having amenorrhea. Testicular suppression (reversible and irreversible)  has been reported. The most common adverse reaction is  myelosuppression. Irreversible BM failure has been reported.  Gastrointestinal side effects reported include nausea/vomiting, diarrhea  and oral mucosa ulceration. Hepatic toxicity has occurred, including  veno-occlusive disease. Acute hypersensitivity reactions occur in about  2.4% of patients, and can include anaphylaxis. Hypersensitivity reactions  were characterized by urticaria, pruritus, and edema. Some patients  exhibited tachycardia, bronchospasm, dyspnea and hypotension that  responded to antihistamines and corticosteroids. Other side effects that  have been reported include skin ulceration or necrosis at injection site,  vasculitis, alopecia, hemolytic anemia, pulmonary fibrosis, and interstitial  pneumonitis.   |
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| Route        | Intravenous                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |  
| Mesna (Mesnex)  Source &  Mesna is a synthetic sulfhydryl (thiol) compound. Mesna contains free  Pharmacology  sulfhydryl groups that interact chemically with urotoxic metabolites of  oxaza-phosphorine derivatives such as cyclophosphamide and ifosfamide. Oral  bioavailability is 50%. Upon injection into the blood, mesna is oxidized to  mesna disulfide, a totally inert compound. Following glomerular filtration,  mesna disulfide is rapidly reduced in the renal tubules back to mesna, the  active form of the drug. Mesna and mesna disulfide are excreted primarily via  the urine.  Formulation  Mesna is available in 2 ml, 4 ml and 100 ml amps containing 100 mg/ml of  and Stability  mesna solution. The intact vials can be stored at room temperature. Mesna  may be further diluted in 5% dextrose or 0.9% NaCl containing solutions.  Diluted solutions are physically and chemically stable for at least 24 hours  under refrigeration.  Supplier Commercially available   |
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| T o xicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Mes na is ge ner all y well t olerate d.                          | Na us ea a n d v o miti n g, hea dac he, diarr hea,                            |                         |
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| ras h, tra nsie nt h y p ote nsi o n a n d aller gic reacti o ns ha ve bee n r e p orte d.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Patie nts                                                         |                                                                                |                         |
| ma y c o m plai n of a bitter taste i n t heir m o ut h d uri n g a d mi nistrati o n.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Mes na ma y                                                       |                                                                                |                         |
| ca use f alse p ositi ve uri ne di pstic k rea di n gs f or ket o nes.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                   |                                                                                |                         |
| D osa ge                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | a n d                                                             |                                                                                |                         |
| A d mi nistrati o n                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Mes na is ge nerall y d ose d at a p pr o xi matel y 2 5          | % of t he c ycl o p h os p ha mi de d ose.                                     |                         |
| It is ge n er all y gi ve n i ntra ve n o usl y pri or t o a n d a gai n at 3, 6 a n d 9 h o urs  f oll o wi n g eac h d ose of c ycl o p h os p ha mi de.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                   |                                                                                |                         |
| R o ute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | I ntr a ve n o us                                                 |                                                                                |                         |
| T hi ote pa ( T hi o ple x                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | b y I m m u ne x) ( T E S P A, T S P A)                           |                                                                                |                         |
| S o urce &  P har mac ol o g y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | T hi ote pa is a cell                                             | c ycle n o ns pecific p ol yf u n cti o nal al k ylati n g a g e nt. It reacts |                         |
| wit h D N A p h os p hate gr o u ps t o pr o d uce cr oss                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | li n ki n g of D N A stra n ds lea di n g                         |                                                                                |                         |
| t o i n hi biti o n of D N A, R N A a n d pr otei n s y nt hesis.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | T hi ote pa is e xte nsi vel y                                    |                                                                                |                         |
| meta b olize d i n t he li ver t o                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | meta b olites t hat retai n acti vit y, pri maril y               |                                                                                |                         |
| triet h yle ne p h os p h or a mi de ( T E P A).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | T he mai n r o ute of eli mi nati o n is via t he                 |                                                                                |                         |
| uri ne, mai nl y a s meta b olites; t he eli mi nati o n halflife of t he t hi ote pa is 2. 5  h o urs, a n d t hat of T E P A is 1 7. 6 h o urs.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                   |                                                                                |                         |
| F or m ulati o n a n d  Sta bilit y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | T hi ote pa is s u p plie d i n si n gle                          | use vials c o nt ai ni n g 1 5                                                 | m g of l y o p hilize d |
| t hi ote pa, 8 0 m g Na Cl a n d 5 0 m g Na H C O 3.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | T he i ntact vials s h o ul d be st ore d                         |                                                                                |                         |
| u n der refri gerati o n a n d pr otecte d fr o m li g ht.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Eac h vial s h o ul d be                                          |                                                                                |                         |
| rec o nstit ute d wit h 1. 5 ml of sterile water f or i njecti o n t o yiel d a c o nce ntrati o n  of 1 0 m g/ ml. F urt her dil uti o n wit h sterile water f or i njecti o n t o a  c o nce ntr ati o n of 1 m g/ ml yiel ds a n is ot o nic s ol uti o n; if lar ger v ol u mes are  desire d f or i ntraca vitar y, i ntra ve n o us i nf usi o n, or perf usi o n t hera p y, t his  s ol uti o n ma y t he n be dil ute d wit h 5 % de xtr ose or 0. 9 % Na Cl c o ntai ni n g  s ol uti o ns. T he 1 0 m g/ ml rec o nstit ute d s ol uti o n is c he micall y sta ble w he n  st ore d i n t he refri gerat or f or u p t o 5 da ys, h o we ver, it is rec o m me n de d t hat  s ol uti o ns be pre pare d j ust pri or t o a d mi nistrati o n si nce t he y d o n ot c o ntai n a  preser v ati ve. Rec o nstit ute d s ol uti o ns s h o ul d be clear t o sli g htl y o pa q ue: t he  s ol uti o ns ma y be filtere d  t hr o u g h a 0. 2 2 micr o n filter t o eli mi nate haze. |                                                                   |                                                                                |                         |
| S u p plier                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | C o m merciall y a vaila ble; ma n ufact ur e d b y I m m u n e x |                                                                                |                         |
| T o xicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | D ose li miti n g t o xicit y is m yel os u p pressi o n.         | T he l e u k oc yte n a dir ma y o cc ur at                                    |                         |
| a n y ti me fr o m 1 0 t o 3 0 da ys.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Ot her t o xicities i ncl u de pai n at t he i njecti o n site,   |                                                                                |                         |
| na usea a n d v o miti n g, a n ore xia, m uc ositis, dizzi ness, hea dac h e, a me n orr h ea,  i nterfer e nce wit h s per mat o ge nesis, a n d de pi g me ntati o n wit h t o pical use.  Aller gic r eacti o ns, i n cl u di n g s ki n ras h a n d hi ves, ha ve bee n re p orte d r ar el y.  Rare cases of a p nea, he m orr ha gic c ystitis, a n d re nal fail ure ha ve occ urre d.  T hi ote pa is m uta ge nic, carci n o ge nic, a n d ter at o g e nic i n a ni mals. Pre g na nc y  cate g or y D.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                   |                                                                                |                         |
| R o ute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | I ntr a ve n o us i nf usi o n.                                   |                                                                                |                         |
| A me n d me nt 5. 1, date d: 0 6/ 1 3/ 2 0 1 8                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | I R B A p pr o val date: 1 0/ 1 1/ 2 0 1 8                        |                                                                                |                         |  
Pr ot oc ol d oc u me nt date: 0 9/ 0 6/ 2 0 1 8  
| M yc o p he n olate m of etil (                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | M                                                                                                                                                                                                                     | M F,                | Cell Ce pt               |                  |                 |     |                    |       |    |         |     |
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| S o urce  &  P har mac ol o g y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | M yc o p he n olate                                                                                                                                                                                                   |                     | m ofetil                 | is               | h y dr ol yze d | t o | m yc o p he n olic | aci d | (  | M P A), | a n |
| i m m u n os u p pressi ve a ge nt.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | M P A i n hi bits                                                                                                                                                                                                     | T- a n d            | B cell pr oliferati o n, | Tcell            |                 |     |                    |       |    |         |     |
| s y nt hesis, a n d a nti b o d y secreti o n b y p ote nt, n o nc o m petiti ve re versi ble i n hi biti o n  of i n osi ne m o n o p h os p hate de h y dr o ge nase (I M P D H) i n t he p uri ne bi os y nt hesis  pat h wa y. I n hi biti o n of I M P D H res ults i n a de pleti o n of g ua n osi ne tri p h os p hate  a n d de o x y g ua n osi ne tri p h os p hate, i m p orta nt i nter me diates i n t he s y nt hesis of  l y m p h oc yte D N A, R N A, pr otei ns a n d gl yc o pr otei ns. Oral f or m ulati o ns of  m yc o p he n olate m ofetil are ra pi dl y a n d e xte nsi vel y a bs or be d w he n gi ve n o n a n  e m pt y st o mac h. F o o d a n d al u mi n u m - a n d ma g nesi u m c o ntai ni n g a ntaci ds  decrease a bs or pti o n of m yc o p he n olate m ofetil. M M F is ra pi dl y h y dr ol yze d t o t he  acti ve meta b olite ( M P A) after oral or i ntra ve n o us a d mi nistrati o n. Free M P A is  c o nj u gate d i n t he li ver b y gl uc ur o n yl tra nsferase t o i nacti ve m yc o p he n olic aci d  gl uc ur o ni de ( M P A G) w hic h is e xcrete d i n t he uri ne a n d feces. Ti me t o pea k  plas ma c o nce ntrati o n is 0. 8 - 1. 3 h o urs, a n d t he mea n eli mi nati o n half life is 1 7. 9  h o urs. E nter o he patic recirc ulati o n of M P A c o ntri b utes t o plas ma c o nce ntrati o ns.  A d mi nistrati o n of c h olest yra mi ne i nterr u pts t he e nter o he patic recirc ulati o n a n d  ca n decrease bi oa v aila bilit y b y as m uc h as 4 0 %. Patie nts wit h re nal i ns ufficie nc y  ha ve i ncrease d plas ma c o nce ntrati o ns of M P A a n d M P A G. Ac ycl o vir a n d  ga ncicl o vir ma y c o m pete wit h M P A G f or re nal t u b ular secreti o n, res ulti n g i n  i ncrease d plas ma c o nce ntrati o ns of b ot h dr u gs. |                                                                                                                                                                                                                       |                     |                          |                  |                 |     |                    |       |    |         |     |
| F or m ulati o n  a n d Sta bilit y                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | M yc o p he n olate m ofetil is c o m merciall y a vaila ble as 2 5 0 m g ca ps ules, 5 0 0 m g  ta blets, 2 0 0 m g/ ml p o w der f or or al s us pe nsi o n, a n d 5 0 0 m g vials of p o w der f or  i njecti o n. |                     |                          |                  |                 |     |                    |       |    |         |     |
| S u p plier                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | C o m merciall y a vaila ble                                                                                                                                                                                          |                     |                          |                  |                 |     |                    |       |    |         |     |
| T o xicities                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | A d verse e ve nts see n i n patie nts ta ki n g                                                                                                                                                                      | m yc o p he n olate | m of etil i ncl u de     |                  |                 |     |                    |       |    |         |     |
| h y perte nsi o n,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | h y p ote nsi o n,                                                                                                                                                                                                    | peri p heral        | e d e ma,                | le u k o pe nia, | a ne mia,       |     |                    |       |    |         |     |
| t hr o m b oc yt o pe nia, h y p o c hr o mic a n e mia, le u k oc yt osis, hea dac h e, i ns o m nia,  dizzi ness, tre m or, a n xiet y, parest h esia, h y per gl yce mia, h y per c h olester ole mia,  h y p o kale mia, h y per k al e mia, h y p o p h os p hate mia, diarr h ea, c o nsti pati o n,  na usea, v o miti n g, a n or e xia, a b d o mi nal pai n, d ys pe psia, uri nar y b ur ni n g or  fre q ue n c y, re n al t u b ular necr osis, he mat uria, i n creas e ser u m cr eati ni ne a n d  B U N, a variet y of i nfecti o ns d ue t o i m m u n os u p pressi o n, ras h, ac ne, o c ular  c ha n ges (catar acts, ble p haritis, keratitis, gla uc o ma, a n d mac ular  a b n or malities) occasi o nal le g cra m ps or p ai n, b o ne pai n, m yal gias, a n d h a n d  cra m ps. I ntra ve n o us i nf usi o ns ha ve b ee n re p ort e d t o ca use t hr o m b osis a n d  p hle bitis. T here ha ve bee n occasi o nal r e p orts of gastr oi ntesti nal he m orr ha ge.  Hi g h d ose t hera p y wit h m yc o p he n olate i n a d ults wit h ps oriasis has bee n  ass ociate d wit h t he f oll o wi n g ne o plas ms: a de n ocarci n o ma of t he breast a n d  c ol o n, basal cell car ci n o ma, car ci n o ma of t he gall bla d der, histi oc ytic  l y m p h o ma, gli o blast o ma m ultif or me, a n d s q ua m o us cell carci n o ma of t he  e pi gl ottis.                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                       |                     |                          |                  |                 |     |                    |       |    |         |     |
| R o ute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | I ntr a ve n o us or oral                                                                                                                                                                                             |                     |                          |                  |                 |     |                    |       |    |         |     |
| A me n d me nt 5. 1, date d: 0 6/ 1 3/ 2 0 1 8                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | I R B A p pr o val date: 1 0/ 1 1/ 2 0 1 8                                                                                                                                                                            |                     |                          |                  |                 |     |                    |       |    |         |     |  
Pr ot oc ol d oc u me nt date: 0 9/ 0 6/ 2 0 1 8