Conditions: B-Lymphoid Malignancies Acute Lymphocytic Leukemia Chronic Lymphocytic Leukemia Non-hodgkin Lymphoma
Keywords: B-Lymphoid Malignancies Relapsed/Refractory Acute lymphocytic leukemia ALL Chronic lymphocytic leukemia CLL Non-Hodgkin lymphoma NHL (CAR).CD19-CD28-zeta-2A-iCasp9-IL15-transduced cord blood natural killer cells CB-NK cells Fludarabine Fludarabine phosphate Fludara Cyclophosphamide Cytoxan Neosar AP1903 Mesna Mesnex
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 49
Healthy volunteers: no

Brief summary:
If you are reading and signing this form on behalf of a potential participant, please note: Any time the words "you," "your," "I," or "me" appear, it is meant to apply to the potential participant.

The goal of this clinical research study is to learn if giving genetically changed immune cells, called CAR-NK cells, after chemotherapy will improve the disease in stem cell transplant patients with relapsed (has returned) and/or refractory (has not responded to treatment) B-cell lymphoma or leukemia. Also, researchers want to find the highest tolerable dose of CAR-NK cells to give to patients with relapsed or refractory B-cell lymphoma or leukemia. The safety of this treatment will also be studied.

This is an investigational study. The making of and infusion of genetically changed NK cells and the drug AP1903 (if you receive it, explained below) are not FDA approved or commercially available for use in this type of disease. They are currently being used for research purposes only. The chemotherapy drugs in this study (fludarabine, cyclophosphamide, and mesna) are commercially available and FDA approved.

Up to 36 patients will take part in this study. All will be enrolled at MD Anderson.

Detailed description:
Objectives:

Primary objective:

To determine the safety and relative efficacy of Chimeric antigen receptors (CAR).CD19-CD28-zeta-2A-iCasp9-IL15-transduced cord blood natural killer (CB-NK) cells in patients with relapsed/refractory CD19+ B lymphoid malignancies.

Secondary Objectives:

1. To assess the overall response rate (complete and partial response rates).
2. To quantify persistence of infused allogeneic donor CAR-transduced CB-derived NK cells in the recipient.
3. To conduct comprehensive immune reconstitution studies.

Arms:
arm 1: On Days -5, -4, and -3, participants receive Fludarabine and Cyclophosphamide. Participants also receive Mesna before and after the cyclophosphamide dose.

On Day 0, participants receive genetically modified NK cells as a cell infusion.

If participant has graft-versus-host disease (GvHD) or cytokine release syndrome after the NK cell infusion, they receive AP1903 by vein and possibly steroids by mouth or by vein.

Interventions: intervention 1: Fludarabine intervention 2: Cyclophosphamide intervention 3: Mesna intervention 4: iC9/CAR.19/IL15-Transduced CB-NK Cells intervention 5: AP1903
Intervention descriptions: intervention 1: 30 mg/m2 by vein on Days -5 to -3. intervention 2: 300 mg/m2 by vein on Days -5 to -3. intervention 3: 300 mg/m2 by vein on Days -5 to -3 before and after the cyclophosphamide dose. intervention 4: Infusion of iC9/CAR.19/IL15-transduced CB-NK cells on Day 0 by vein.

Starting dose: 10E5 intervention 5: If participant has graft-versus-host disease (GvHD) or cytokine release syndrome after the NK cell infusion, they will receive AP1903 0.4 mg/kg administered as an intravenous infusion.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
      
 
 
 
 
 
 
 
MD Anderson IND Sponsor Cover Sheet 
Protocol ID 
2016-0641 
Protocol Title 
Dose Escalation Study Phase I/II of Umbilical Cord Blood-Derived 
CAR-Engineered NK Cells in Conjunction with Lymphodepleting 
Chemotherapy in Patients with Relapsed/Refractory B-Lymphoid 
Malignancies 
 
Protocol Phase: 
Phase I/II 
Protocol Version 25 
Version Date 
26 August 2020 
Protocol PI 
Loretta Nastoupil, MD 
Department 
Stem Cell Transplantation and Cellular Therapy 
IND Sponsor 
MD Anderson Cancer Center 
IND # 
17321 
 
 
 
 
 
 
 
 
 
 
 


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Dose Escalation Study Phase I/II of Umbilical Cord Blood-Derived  
CAR-Engineered NK Cells in Conjunction with Lymphodepleting 
Chemotherapy in Patients with Relapsed/Refractory  
B-Lymphoid Malignancies 
 
 
Institution Study Number: 
2016-0641 
Financial Support: 
Takeda Pharmaceutical Company 
IND Sponsor: 
The University of Texas MD Anderson Cancer Center 
IND Number: 
17321 
Principal Investigator: 
Loretta Nastoupil, MD 
The University of Texas MD Anderson Cancer Center 
Stem Cell Transplantation and Cellular Therapy Department 
1515 Holcombe Blvd, Unit 0432 
Houston, TX 77030 
Telephone: 713.792.2860 
Fax: 713.794.5656 
lnastoupil@mdanderson.org 
Co-Chair: 
Chitra Hosing, MD1 
Collaborators: 
Richard E. Champlin, MD1  
Nitin Jain, MD2 
Michael J. Keating, MD2 
Partow Kebriaei, MD1 
Sajad J. Khazal, MD3 
Kris M. Mahadeo, MD3 
Rohtesh Mehta, MD1  
Yago Nieto, MD, PhD1 
Amanda L. Olson, MD1 
Betul Oran, MD1 
Demetrios Petropoulos, MD3 
Uday Popat, MD1 
Muzaffar H. Qazilbash, MD1  
Peter F. Thall, PhD4 
Philip A. Thompson, MD2 Michael 
Wang, MD5 
William G. Wierda, MD2 
 
 
1 The University of Texas MD Anderson Cancer Center, Stem Cell Transplantation and Cellular Therapy Department 
2 The University of Texas MD Anderson Cancer Center, Leukemia Department 
3 The University of Texas MD Anderson Cancer Center, Division of Pediatrics 
4 The University of Texas MD Anderson Cancer Center, Biostatistics Department 
5 The University of Texas MD Anderson Cancer Center, Lymphoma/Myeloma Department 
 


 
 
 
 
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Table of Contents 
 
 
1.0     Objectives ..................................................................................................................................... 3 
2.0     Background .................................................................................................................................. 3 
3.0     Patient Eligibility ......................................................................................................................... 14 
4.0      Treatment Plan ........................................................................................................................... 15 
5.0 
Evaluation During Study............................................................................................................. 18 
6.0      Background Drug Information .................................................................................................... 22 
7.0      Criteria for Removal from the Study ........................................................................................... 24 
8.0 
Statistical Considerations ........................................................................................................... 25 
9.0      Adverse Events and Reporting Requirements ........................................................................... 27 
10.0 
References ................................................................................................................................ 32 
Appendix A. - CARTOX: CAR T-Cell Therapy Toxicity Assessment and Management ......................... 39 
Appendix B. -  CARTOX: CTCAE Grading of Common Organ Toxicities .............................................. 43 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
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Protocol Body  
1.0  Objectives 
 
Primary objective: 
To determine the safety and relative efficacy of CAR.CD19-CD28-zeta-2A-iCasp9-IL15-
transduced CB-NK cells in patients with relapsed/refractory CD19+ B lymphoid malignancies. 
 
Secondary Objectives: 
1. To assess the overall response rate (complete and partial response rates). 
2. To quantify persistence of infused allogeneic donor CAR-transduced CB-derived NK cells in 
the recipient. 
3. To conduct comprehensive immune reconstitution studies. 
2.0 Background 
The aim of this clinical trial is to investigate novel immunotherapeutic strategies, using 
engineered natural killer (NK) cells to improve the tumor-free survival of patients with relapsed 
or refractory CD19+ B-cell malignancies. There are an annual average of 69,740 new cases of 
non-Hodgkin lymphoma (NHL), 15,680 new cases of chronic lymphocytic leukemia (CLL) and 
6070 new cases of acute lymphoblastic leukemia (ALL) in the United States, with estimated 
annual death rates of 19,020, 4580 and 1,430 respectively (http:/www.cancer.org). Overall 
survival (OS) is determined largely by disease stage at presentation and response to 
chemotherapy. Standard therapy for patients who relapse following frontline therapy is 
allogeneic hematopoietic stem cell transplantation (HSCT). The expected OS for patients in 2nd 
complete remission is 25% based on chemotherapy-sensitivity at the time of HSCT. Thus, there 
is an urgent and unmet need to develop new therapies for patients with advanced B-lineage 
malignancies, especially because relapse after allogeneic HSCT is usually fatal. 
 
Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the 
United States, accounting for 25% of all leukemias. There are more than 15,000 new cases of 
CLL and 4,500 deaths from CLL every year in the United States. The natural history of the 
disease is diverse. Patients with only lymphocytosis have a median survival greater than of 10 
years, whereas those with evidence of marrow failure manifested by anemia or 
thrombocytopenia have a median survival of only 2-3 years. Since no treatment has been 
shown to be curative, nor is there objective evidence that a specific treatment prolongs survival, 
treatment is delayed.1The NCI-sponsored CLL Working Group proposed the following 
indications for initiating treatment: 1) weight loss of more than 10% over the preceding 6 
months; 2) extreme fatigue attributable to progressive disease; 3) fever or night sweats without 
evidence of infection; 4) worsening anemia (Rai stage III) or thrombocytopenia (Rai stage IV); 
5) massive lymphadenopathy (>10 cm) or rapidly progressive lymphocytosis (lymphocyte 
doubling time <6 months); or 6) prolymphocytic or Richter’s transformation. Current treatments 
for newly diagnosed CLL include chemotherapy and antibody therapy either alone or in 
combination.1  A variety of novel approaches such as targeted therapy using ibrutinib for treating 
CLL are being developed,2;3 but the disease is not yet curable. Moreover, even after complete 
responses, immunological abnormalities and minimal residual disease remain in most patients. 
Ultimately, chronic immunosuppression resulting in infectious complications occurs in 80% of 
CLL patients and is a major cause of mortality. Allogeneic stem cell transplantation may be 
curative in some patients with CLL, but success has been limited, primarily due to the high 


 
 
 
 
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incidence of mortality and morbidity associated with the procedure.
4 Non-myeloablative allogeneic 
transplant regimens hold promise, but patient eligibility is limited by availability of matched 
sibling donors. 
 
Historically the initial treatment of patients with CLL requiring treatment has been with an 
alkylating agent, particularly chlorambucil, alone or in combination with a corticosteroid. The 
overall response rate has been 50-70%; however, the complete remission rates are low (5-
20%). Newer agents like purine analogues, particularly fludarabine, have higher response rates 
as initial treatment.5  randomized trials comparing alkylating agent-based therapy with single-
agent fludarabine have shown a higher complete response rate and longer disease-free 
survival with the nucleoside analogue, but have not shown a survival advantage.5;6  Fludarabine 
was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with 
B cell CLL who have not responded to or progressed during treatment with at least one 
alkylating agent-based regimen. 
 
Combination regimens such as cyclophosphamide, fludarabine and Rituximab have been 
shown to improve response rates,7 but these regimens are highly immunosuppressive, and 
long-term benefit has not been demonstrated. Ibrutinib is a covalent inhibitor of Bruton's 
tyrosine kinase (BTK), a member of the TEC tyrosine kinase family8 and a key enzyme in the B-
cell receptor signaling pathway. Ibrutinib as monotherapy, as well as in combination with 
immunotherapy or chemotherapy, is a very effective therapy for lymphoid malignancies 
including CLL.2;9  However, outcomes after ibrutinib failure are dismal, with only a 3.1 month 
survival after drug discontinuation.10 
 
Acute Lymphoblastic Leukemia. Allogeneic HCT is a curative approach for a select  group 
of patients with ALL. Overall survival (OS) ranges from 30%-60% depending on the patients 
disease stage and risk profile at time of transplant.11;12  Increasingly, minimal residual disease 
(MRD), both before and after HCT, is becoming an important predictor for relapse.
13  In a series 
of 149 ALL patients transplanted in remission at MD Anderson Cancer Center, patients with 
MRD, measured by multi-parameter flow cytometric immunophenotyping (FCI) with a 
sensitivity of 0.01%, present at time of HCT had a shorter PFS compared to patients who 
were MRD negative, 28% vs. 47%, p=.08 (4). Furthermore, among 135 patients who had 
MRD measured following HCT, 20 became positive for MRD, and 18 of these patients 
developed overt hematologic relapse within a median of 3.8 months.14 Of note, among 32 
patients with overt relapse following HCT, 41% did NOT have preceding MRD, suggesting 
that positive MRD post HCT essentially confirms eventual relapse, but negative MRD post 
HCT in a high-risk patient does not preclude relapse. Our findings corroborate similar 
published studies.15;16  Patients transplanted beyond second remission routinely have a 
significantly lower PFS and OS rates. In our study of 97 patients (CR1 51, CR2 29, others 17) 
treated with busulfan and clofarabine chemotherapy conditioning following a matched sibling 
(MSD) or matched unrelated donor (MUD) transplant, patients in CR1 had a significantly 
better disease free survival (DFS) compared with others. For patients in CR1, the 2-yr DFS 
rate was 61% with 9/51 patients relapsing at median 9 months, the 2-yr DFS rate was 40% for 
CR2, with 10/29 relapsing at median 3 months, and for patients with more advanced disease, 
the 2-yr DFS rate was 33% with 3/17 progressing at a median of 3 months. Data from the 
Center for International Blood and Marrow Transplant Research (CIBMTR) corroborate our 
findings. Between 1996 and 2001, in patients less than 20 years-old, OS ranges from 25% for 
patients transplanted beyond first remission to 50% for sibling transplants in first remission. 
Similarly, in adult patients, greater than 20 years-old, the best outcome is noted in sibling 
transplants done in first remission with OS of 60%, as compared to 35% if transplants are 


 
 
 
 
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2
2
performed beyond CR1 (CIBMTR Registry). 
No effective treatment options exist for patients who relapse following HCT. Multiple published 
series report less than 10% survival for these patients, regardless of the treatment modality used, 
with a median survival of 2-3 months.11-17  To date, the most common strategy employed to 
reduce relapse rates after HCT has usually involved some form of immune manipulation, 
ranging from donor lymphocyte infusion (DLI) to second transplant.18-20  However, although it has 
been consistently shown that patients with B-ALL who develop graft-versus-host-disease (GVHD) 
have less risk for relapse, DLI has not shown appreciable efficacy in this patient 
population; remission rates have been less than 10%, and have been associated with a high 
incidence of GVHD. Of note, the best responses to DLI in ALL occur when the DLI is 
administered prophylactically to prevent relapse;20 this approach has been demonstrated 
in pediatric patients but no data for prophylactic DLI has been reported in adults. Thus, 
there is an unmet need for effective therapy for ALL patients at high risk for relapse 
following allogeneic HCT, with high risk defined as positive MRD and/or disease beyond 
first complete remission. 
 
Non-Hodgkins Lymphoma (NHL). In the United States, B cell lymphomas represent 80-85% 
of cases reported. In 2013 approximately 69,740 new cases of NHL and over 19,000 deaths 
related to the disease were estimated to occur. Non-Hodgkin lymphoma is the most prevalent 
hematological malignancy and is the seventh leading site of new cancers among men and 
women and account for 4% of all new cancer cases and 3% of deaths related to cancer (SEER 
2014). Diffuse Large B cell Lymphoma: Diffuse large B cell lymphoma (DLBCL) is the most 
common subtype of NHL, accounting for approximately 30% of NHL cases. There are 
approximately 22,000 new diagnoses of DLBCL in the United States each year. First line 
therapy for DLBCL typically includes an anthracycline-containing regimen with rituximab.  The 
first line objective response rate and the complete response (CR) rate to R-CHOP (rituximab, 
cyclophosphamide, doxorubicin, vincristine, and prednisone) is approximately 80% and 50% 
respectively. However, approximately one-third of patients have refractory disease to initial 
therapy or relapse after R-CHOP.24 For those patients who relapse after response to first line 
therapy, approximately 40-60% of patients can achieve a second response with additional 
chemotherapy. For patients who are young and fit, the goal of second line therapy is to achieve 
a response that will make the patient eligible for autologous stem cell transplant (ASCT). The 
standard of care for second-line therapy for transplant-eligible patients includes rituximab and 
combination chemotherapy such as RICE (rituximab, ifosfamide, carboplatin, and etoposide) or 
RDHAP (rituximab, dexamethasone, cytarabine, and cisplatin). In a large randomized trial of 
RICE versus RDHAP in transplant-eligible patients with DLBCL (the CORAL study) 63% of 
patients achieved an objective response to either regimen with a 26% CR rate. Patients who 
respond to second line therapy and who are considered fit enough for transplant receive 
consolidation with high-dose chemotherapy and ASCT. This combination can cure 
approximately 50% of transplanted patients.25  Patients who fail ASCT have a very poor prognosis 
and no curative options. The majority of second line patients are not eligible for ASCT due to 
chemotherapy-refractory disease, age, or comorbidities such as heart, lung, liver, or kidney 
disease. Transplant-ineligible salvage patients do not have a curative option available to them. 
There is no standard definition of relapse/refractory DLBCL. This trial will enroll patients with 
chemo-refractory lymphoma, as evidenced by failure to achieve even a transient or partial 
response to prior biologic and combination chemotherapy or by early recurrence after ASCT. 
 
Transformed Follicular Lymphoma (TFL). Follicular lymphoma (FL), a B cell lymphoma, is the 
most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% 


 
 
 
 
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of all NHLs. Some patients with FL will transform (TFL) histologically to DLBCL which is more 
aggressive and associated with a poor outcome. Histological transformation to DLBCL occurs at 
an annual rate of approximately 3% for 15 years with the risk of transformation continuing to 
drop in subsequent years. The biologic mechanism of histologic transformation is unknown. 
Initial treatment of TFL is influenced by prior therapies for follicular lymphoma but generally 
includes anthracycline-containing regimens with rituximab to eliminate the aggressive 
component of the disease (NCCN practice guidelines 2014). Treatment options for 
relapsed/refractory TFL are similar to those in DLBCL. Given the low prevalence of these 
diseases, no large prospective randomized studies in these patient populations have been 
conducted.26 Patients with chemotherapy refractory disease have a similar or worse prognosis 
to those with refractory DLBCL. 
 
In summary, subjects who have refractory, aggressive B lymphoid malignancies have a major 
unmet medical need and novel treatments are warranted in these populations. 
 
NK cells: 
 
Natural killer (NK) cells are an important component of the graft-versus-leukemia (GVL) response, 
which is critical to preventing relapse after HSCT.33;34   Each mature NK cell expresses a wide array 
of activating and inhibitory killer immunoglobulin-like receptors (KIRs), which are 
specific for different HLA class-I molecules.35-37 The ability of NK cells to recognize and kill 
malignant cells is governed by complex and poorly understood interactions between inhibitory 
signals resulting from the binding of inhibitory KIRs with their cognate HLA class-I ligands, and 
activating signals from activating receptors.33;37;38  NK cell responses are mediated by two 
major effector functions: direct cytolysis of target cells and production of chemokines and 
cytokines. Through the latter mechanism (e.g., interferon-γ), NK cells participate in the 
shaping of the adaptive T cell response, possibly by a direct interaction between naïve T 
cells and NK cells migrating to secondary lymphoid compartments from inflamed peripheral 
tissues and by an indirect effect on dendritic cells (DC).39;40 
 
GMP-grade NK cell expansion from cord blood. Previous studies have largely used freshly 
obtained peripheral blood NK cells. The low number of circulating peripheral blood NK cells 
severely limits their therapeutic utility. We have developed a system for ex vivo expansion of 
NK cells from cord blood (CB), which reliably generates clinically relevant doses of GMP grade 
CB-NK cells for adoptive immunotherapy, using GMP-grade K562-based artificial antigen- 
presenting cells (aAPCs) expressing membrane bound IL-21, 4-1BB ligand, CD64 (FcγRI) and 
CD86 (clone 9.mbIL21).41  Cord blood is a novel, attractive source of NK cells for cellular immune 
therapy. The cells are already collected, stored and immediately available. The cord blood 
donor can be optimally selected for HLA type, KIR gene expression and other factors. The 
methodology to generate CB NK cells has been approved by the FDA.42 Our current protocol 
yields a mean NK expansion of 3127 fold (range, 1640 – 4931 fold) (Fig 1A), with very few 
CD3+ cells (mean, 4.50 x 106) (Fig 1B). 
 


 
 
 
 
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Fig 1. Expansion, phenotype and anti-leukemia activity of ex vivo expanded CB-NK cells in vitro. A. 
Culture of CB-NK cells: unselected fresh or frozen CB-MNCs were cultured for 7 days in a GP500 bioreactor 
with IL-2 alone or with IL-2 and clone 9.mbIL21 (antigen presenting cell, APC). Cells were immunomagnetically 
CD3-depleted on day 7 and re-cultured in the same conditions for an additional 7 days. On day 7, cells were 
again CD3-depleted and subjected to phenotypic and functional studies. B. Purity of NK product during 
expansion. C. APC-expanded CB-NK ells maintained Eomeshi and T-bethi phenotype. D. Representative 
histograms of NK receptor expression on ex-vivo expanded CB vs. PB NK cells. E. APC-expanded CB-NK 
cells demonstrate robust cytotoxicity against AML cell targets. 
 
 
Fig. 2. Adoptively transferred CB-NK cells home to lymphoid and non-lymphoid tissues in 
NOD/SCID/IL2Rγc (NSG) mice. Intravenous infusion of 10 million CB-NK cells into lymphocyte-deficient NSG 
mice leads to homing of human NK cells to multiple lymphoid and non-lymphoid organs. (A, B) Flow cytometric 
analysis of single cell suspensions (live cells) for the detection of CB-NK cells. (A) Human CD45+ and (B) 
human CD56+ cells, gated on human CD45+ cells shown in (a). Ten recipients were analyzed at 20 hours 
after adoptive transfer. (C, D) Percentages of human CD45+CD56+ cells of total life single cells of each 
indicated organ. 
 
Functional phenotype of ex vivo-expanded CB-NK cells and their cytotoxic activity 
against myeloid leukemia targets. The expanded CB-NK cells display the full array of 
activating and inhibitory receptors, continue to strongly express eomesodermin (Eomes) and 
T-bet (Fig 1C-D), two factors necessary for NK cell maturation and activation,43;44  lyze 
myeloid target cells in a dose-dependent manner (FIG. 1E) and upon adoptive transfer into 
non-obese diabetic severe combined immunodeficient-gamma null (NSG) mice, could home 
to the bone marrow, liver, spleen and multiple lymphoid tissues (FIG. 2). 


 
 
 
 
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Fig. 3. Flow cytometric analysis to determine the persistence and phenotype of adoptively infused CB-
NK cells in a patient with myeloma following high dose chemotherapy and autologous stem cell 
rescue. Using flow cytometry and HLA class I-specific antibodies the source of recovering NK cells (donor vs. 
recipient) is identified. In this case, the donor is HLA-Bw6+ve while the recipient is HLA-Bw6 negative. CB-
derived NK cells are detectable in the peripheral blood of the recipient up to 21 days post-infusion. 
 
 
MD Anderson experience with infusion of ‘off-the-shelf’ ex vivo expanded CB-derived NK 
cells. At MD Anderson Cancer Center, we have initiated a phase 1, first-in-human clinical trial 
with off-the-shelf allogeneic CB-NK cells in combination with high-dose chemotherapy and 
autologous hematopoietic stem cell transplant in patients with multiple myeloma 
(NCT01729091). To date we have treated 32 patients in three cohorts: 3 received 5 x10e6 
expanded CB-NK cells/kg, 3 received 1 x10e7 CB-NK cells/kg, 3 received 5 x10e7 CB-NK 
cells/kg and 23 have received 1 x10e7 CB-NK cells/kg. Thus far the infusions of CB-NK cells 
appear to be well-tolerated without any toxicity. With follow-up times ranging from 1-24 months, 
there have been no cases of delayed recovery of neutrophil or platelet counts or GVHD. In the 
third and fourth cohorts we have detected CB-derived NK cells up to three weeks post-infusion  
(Fig. 3). 
 
In a second phase I study (NCT01823198), we treated 18 patients who received HLA-
compatible related or unrelated donor HSCT with increasing doses of ex vivo expanded NK 
cells (1 x10e6-1x10e8/kg) from third party (haploidentical relative or unrelated CB unit) to 
improve transplant outcomes. To date, there have been no major toxicities, no cases of graft 
failure and no increase in the incidence of GVHD. These studies confirm the safety and 
feasibility using ex vivo expanded CB-NK cells as a source of off-the-shelf immunotherapy. 
 
Genetic modification of CB-derived NK cells to enhance their activity against  leukemia. 
Chimeric antigen receptors (CARs) have been used extensively to redirect the specificity of T 
cells against leukemia45-47 with dramatic clinical responses in patients with acute lymphoblastic 
leukemia (ALL).48-50  
 These infusions have been primarily restricted to the autologous setting 
because activated T cells from an allogeneic source are likely to increase the risk of GVHD. 
Here, we propose to test the safety and efficacy of engineered CB-derived NK cells, as an 
alternative to T cells, for the immunotherapy of B-lymphoid malignancies. CB-derived NK cells 
have multiple potential advantages over T cells: (i) allogeneic NK cells should not cause GVHD, 
as predicted by observations in murine models, as well as patients with leukemia and solid 
malignancies treated with haploidentical or CB-derived NK cells;51-53 (ii) mature NK cells have a 
limited life-span of a few weeks, allowing for antitumor activity while reducing the probability of 
long-term adverse events such as prolonged cytopenias caused by on-target/off-tumor toxicity 
to normal tissues, or the risk of malignant transformation; (iii) Unlike T-cells, NK cells will also 
have activity through their native receptors to kill antigen-negative target cells, potentially 
preventing a mechanism of immune escape; (iv) the generation of an autologous T cell product 
for each patient is logistically cumbersome and restrictive.33;52 The use of frozen, off-the-shelf 


 
 
 
 
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CB units stored in the large global cord blood bank inventory for the generation of NK cells has 
the potential for widespread scalability that would not be possible with autologous peripheral  
blood-derived T or NK cell products. 
 
Thus, to improve the persistence and anti-leukemic potency of frozen and ex vivo expanded 
CB-NK cells, we genetically modified them with a retroviral vector, iC9.CAR19-CD28-zeta-2A-
IL15 (iC9/CAR.19/IL15), that (i) incorporates the gene for CAR-CD19 to redirect their specificity 
to CD19; (ii) ectopically produces IL-15, a cytokine crucial for NK cell survival and 
proliferation,54-55 and (iii) expresses a suicide gene, based on inducible caspase-9 (iC9), that can 
be pharmacologically activated to eliminate transgenic cells as needed. Our preliminary data 
show that CB-NK cells can be stably transduced to express the CAR molecule (Fig 4A). Using 
a standard 51Cr-release assay, we found that iC9/CAR.19/IL15-transduced CB-NK cells had 
specific cytotoxic activity against CD19+ Raji cells and primary CLL cells (n=18; Fig 4B). The 
NK-CAR and non-transduced NK cells showed equal effector function against K562 cells, 
indicating that the genetic modification of CB-NK cells did not alter their intrinsic cytotoxicity 
against NK-sensitive targets (Fig 4B). 
 
 
 
Fig. 4. Antileukemic function of CB-NK cells transduced with CAR19-CD28-zeta-2A-IL15 vector. (A) 
Transduction efficiency (85%) of CB NK cells (bottom panel) compared to non-transduced NK cells (top panel). 
Transduction is stable. (B) CAR-NK cells are more efficient at killing CD19+ Raji tumors and primary CLL 
compared to non-transduced (NT) ex vivo expanded and activated NK cells  with equal effector function against 
K562 cells. P < 0.001 (iC9/CAR.CD19/IL15 + Raji vs NT-NKs + Raji); P < 0.001 (iC9/CAR.CD19/IL15 + CLL 
vs NT-NKs + CLL); P=ns (iC9/CAR.CD19/IL15 + K562 vs NT-NKs + K562). 
 
 
We next evaluated the trafficking and persistence of iC9/CAR.19/IL15-modified CB-NK 
cells in vivo, using a NSG mouse Raji xenograft model. NT and iC9/CAR.19/IL15-
transduced CB-NK cells were infused in mice engrafted with Raji cells. As shown in 
Fig. 5A, iC9/CAR.19/IL15+ CB-NK cells homed to the spleen, liver and bone marrow 
(sites of tumor infiltration), while CAR.CD19+ CB-NK cells without the IL-15 gene in the 
construct, as well as NT CB-NK cells were barely detectable in the tumor sites. 
 


 
 
 
 
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Fig. 5. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB NK cells. 
(A) iC9/CAR.19/IL15-tranduced eGFP-FFLuc-labeled CB-NK cells home to sites of disease (liver, spleen, 
bone marrow [BM]) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells. (B) Infusion of 
iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with luciferase-labeled Raji cells results in 
tumor eradication, as evidenced by in vivo bioluminescence imaging. Colors indicate intensity of luminescence 
(red, highest; blue, lowest). (C). The in vivo antitumor activity of a single dose of iC9/CAR.19/IL15-transduced 
CB NK  is significantly better than that of CB-NK cells that were either not transduced or transduced with a 
CAR.CD19 construct lacking IL-15. P= 0.001 (iC9/CAR.CD19/IL15 + Raji vs NT-NKs + Raji); P= 0.044 
(iC9/CAR.CD19/IL15 + Raji vs CAR.CD19 + Raji); P= 0.006 (CAR.CD19 + Raji vs NT-NKs + Raji) P= 0.182 
(NT-NKs + Raji vs Raji alone). 
 
iC9/CAR.19/IL15-tranduced CB-NK cells exert enhanced anti-tumor activity in vivo. 
To study the in vivo antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells, we 
injected NSG mice with FFLuc-labeled Raji cells at 2 × 105/mouse. On the same day, 
mice received one i.v infusion of control NT, CAR.19 or iC9/CAR.19/IL15-transduced CB-
NK cells (10 x 16/mouse). Tumor growth was monitored by measuring changes in tumor 
bioluminescence over time. As shown in Fig. 5B, tumor bioluminescence increased 
rapidly in mice engrafted with Raji cells and treated with control NT CB-NK cells. By 
contrast, infusion of either CAR.19+ or iC9/CAR.19/IL15+ CB-NK cells resulted in 
significant prolongation of survival compared to the effect of NT CB-NK cells (P=0.006 
and P=0.001, respectively). Notably, iC9/CAR.19/IL15+ CB-NK cells controlled tumor 
expansion and prolonged survival (Fig. 5C) significantly better than the CAR.CD19 
construct lacking the IL-15 gene, underscoring the important contribution of IL-15 to 
enhanced antitumor activity. 
 
 


 
 
 
 
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iC9/CAR.19/IL15-transduced CB-NK cells do not show in vitro or in vivo signs of 
autonomous or dysregulated growth. To investigate the possibility that the IL-15 gene 
in the vector may result in autonomous or dysregulated growth of transduced CB-NK 
cells, we cultured iC9/CAR.19/IL15-transduced CB-NK cells in complete Serum-free 
Stem Cell Growth Medium (SCGM) without the addition of exogenous IL-2 or clone 
9.mbIL21 stimulation for 42 days (n=5). Viable cells were enumerated and passaged 
every three days by replacing media with fresh complete SCGM. As shown in Fig 6A, 
the iC9/CAR.19/IL15-transduced CB-NK cell cultures did not show any signs of 
abnormal growth over 6 weeks, after which, the cells stopped expanding. Karyotyping 
performed on iC9/CAR.19/IL15-transduced CB NK cells cultured for up to 17 weeks 
(n=7) failed to detect any chromosomal alterations (data not shown). We also performed 
chromosome and SNP microarray analyses on paired CB-NK cells (n=6) before (at 
baseline) and up to 22 weeks after CAR-transduction and ex vivo expansion, and did not 
observe any evidence of genetic instability (data not shown). With a follow-up exceeding 
10 months, we did not observe any evidence of autonomous growth or leukemic 
transformation in mice treated with iC9/CAR.19/IL15 or CAR.19-transduced CB-NK cells. 
Histopathologic examination did not reveal any lymphocytic infiltration, proliferation or 
lymphoma in any tissue of these mice. The rudimentary lymphoid tissues of the spleen 
and lymph nodes were free of lymphocytes in all NSG mice from both groups of animals 
(Fig 6B), nor was there any lymphocytic infiltration or proliferation in the bone marrow of 
these mice. Hematologic tests indicated normal numbers of white blood cells and 
lymphocytes, and no evidence of lymphocytic leukemia in both groups of mice. 
 
 
Fig. 6. IL-15-transduced CB-NK cells do not show signs of autonomous or dysregulated growth. (A) 
iC9/CAR.19/IL15-transduced CB NK cells stop expanding within 6 weeks of in vitro culture with no evidence 
of autonomous growth. (B) Photomicrographs of mesenteric lymph nodes show vestigial lymphoid tissue 
with no lymphocytes in any experimental mice, which is typical of NSG mice. Images of the spleen show 
rudimentary periarteriolar lymphoid tissue devoid of lymphocytes (black arrows) and is surrounded by 
hematopoietic tissue composed of various stages of erythroid and myeloid series cells, including 
megakaryocytes and hemosiderin-laden macrophages. Bone marrow contains normal hematopoietic cells 
and no abnormal lymphocytes. H&E stain, magnification x200. Slides from two representative groups of 
NSG mice treated with iC9/CAR.19/IL15-transduced CB NK cells. 
 
IL-15 production by CAR-transduced NK cells 
To verify that iC9/CAR.19/IL15+ CB-NK cells can produce IL-15, we cultured control NT 
CB-NK and iC9/CAR.19/IL15+ CB-NK lymphocytes in triplicates in the presence or 
absence of CD19+ CLL B cells and collected culture supernatants to measure IL15 
release after 24, 48 and 72 hours of culture. As shown in Fig. 7, IL15 was undetectable 
in supernatants collected from non-transduced CB-NK cells cultured alone or with CLL 
targets. By contrast, iC9/CAR.19/IL15+ CB-NK cells produced small amounts of IL15 in 


 
 
 
 
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the absence of antigen stimulation [average 15.05 pg/mL/106 cells (range 6.2 – 
23.47pg/mL)], which significantly increased with antigen stimulation [average 27.61 
pg/mL/106 cells (range 15.82 – 38.18 pg/mL)] (P=0.02). We next examined the ability of 
iC9/CAR.19/IL15-transduced NK cells to produce IL-15 in vivo in NSG mice engrafted 
with Raji cells. Serum levels of IL-15 levels the height of NK cell expansion (2 weeks 
post expansion) were 40-50 pg/mL, and equivalent to levels detected in the supernatant 
of cultured cells. 
 
 
Fig. 7. IL-15 production and phenotype of iC9.CAR.19.CD28.CD3ζ.IL15-transduced CB NK cells. (A) 
iC9. CAR.19.CD28.CD3ζ.IL15-transduced CB NK cells produce IL-15 in response to antigenic stimulation in 
vitro 
 
Exogenous recombinant human IL-15 (RhIL-15) has been used in the clinical setting. In a  
recent phase 1 study in patients with metastatic melanoma or renal cell carcinoma, bolus 
infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-15 were administered for 12 consecutive days 
to patients with metastatic malignant melanoma or metastatic renal cell cancer.57 RhIL-15 was 
shown to activate NK cells, monocytes, γδ, and CD8 T cells. The 3.0-, 1.0-, and 0.3-μg/kg per 
day doses resulted in a maximum serum concentration (Cmax) of 43,800 ± 18,300, 15,900 ± 
1,900, and 1,260 ± 350 pg/mL, respectively. Dose-limiting toxicities observed in patients 
receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and 
elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-
tolerated dose We do not anticipate severe toxicity releated to IL-15 release by 
iC9/CAR.19/IL15-transduced CB-NK cells, as the levels of IL-15 produced by the transduced 
NK cells are on average 2-3 logs lower than that achieved in the clinical trial of exogenous IL-15 
treatment.57 
iC9/CAR.19/IL15+ CB-NK cells are eliminated after activation of the suicide gene by 
exposure to a small-molecule dimerizer. To counteract the possibility of excessive toxicity 
mediated by the release of inflammatory cytokines by transduced CB-NK cells or uncontrolled 
NK cell growth, we incorporated a suicide gene based on the inducible caspase-9 gene in our 
construct.56   As shown in Fig. 8A, the addition of as little as 10 nM of a small molecule dimerizer 
to cultures of iC9/CAR.19/IL15-transduced CB-NK cells induced apoptosis/necrosis of 60% of 
transgenic cells within 4 hours as assessed by annexin-V and 7AAD staining but had no effect 
on the viability of NT CB-NK cells. The suicide gene was also effective in vivo. Mice were 
engrafted i.v. with Raji tumor cells and treated with iC9/CAR.19/IL15-transduced CB-NK cells. 
Administration of the small-molecule dimerizer AP1903 (50 µg, i.p. 2 days apart) 10-14 
days after NK cells had localized and expanded at different tumor sites later (Fig. 8B, left 
panel), resulted in a striking reduction in iC9/CAR.19/IL15-transduced CB-NK cells in the 


 
 
 
 
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blood and tissues of the treated mice (Fig. 8B, right panel), indicative of in vivo 
elimination of the transgenic cells. 
 
 
 
Fig. 8. Activation of the inducible caspase-9 suicide gene eliminates iC9/CAR.19/IL15+ CB-NK cells. (A) 
The addition of 10 nM of AP1903 to cultures of iC9-CAR-IL15+ CB-NK cells induced apoptosis/necrosis of 
transgenic cells (bottom right panel) within 4 hours as assessed by annexin-V-7AAD staining. NT, non-
transduced CB-NK cells; CAR, iC9/CAR.19/IL15-transduced NK cells; (B) NSG mice engrafted i.v. with Raji 
cells, and infused with iC9/CAR.19/IL15+ CB-NK cells were treated 10-14 days later with two doses of the 
AP1903 dimerizer (50 µg) i.p. two days apart. iC9/CAR.19/IL15-expressing NK cells were substantially 
reduced in all organs tested 3 days later. 
 
 
Clinical trial to evaluate the safety and efficacy of CB-NK cells transduced with 
iC9/CAR.19/IL15 in patients with relapsed/refractory B-lymphoid malignancies. 
 
This is a Phase I/II dose-escalation trial to evaluate the safety and relative efficacy of 
iC9/CAR.19/IL15-transduced CB-NK cells in patients with relapsed/refractory B-
lymphoid malignancies (ALL, CLL, NHL). This clinical study will capitalize on the 
synergistic antitumor activity produced by CAR CB-NK cells and the favorable 
lymphopenic environment induced by our lymphodepleting regimen.58.59  Thus, patients 
will be treated with cyclophosphamide at a dose of 300 mg/m2 /day for 3 days. 
Escalating doses of iC9/CAR.19/IL15-transduced CB-NK cells (105/kg-107/kg) will be 
infused once, on day 0, to determine the highest dose at which iC9/CAR.19/IL15-
transduced CB-NK cells can be safely infused into patients with relapsed/refractory B-
lymphoid malignancies, as defined by standard NCI toxicity criteria. A CB unit matched 
at 4/6, 5/6, or 6/6 HLA class I (serological) and II (molecular) antigens with the patient 
will be used for CB-NK expansion and CAR transduction. The CB units will be obtained 
from the MD Anderson cord blood bank. 
 
To gain insight into the persistence, functionality and antileukemic potential of adoptively 
transferred iC9/CAR.19/IL15-transduced CB-NK cells, we have planned a series of 
phenotypic and functional assays. We will evaluate the magnitude of expansion and 
duration of persistence for adoptively infused genetically-modified NK cells in serially 
acquired PB samples by Q-PCR, using a primer pair that specifically amplifies the 
unique CAR transgene with sensitivity to detect 1/10,000 CAR+ NK cells. If there are 
sufficient numbers of circulating NK cells we will quantify by flow cytometry using a mAb 
specific against the CH2-CH3 region of iC9/CAR.19/IL15 with sensitivity to detect 
1/1,000 CAR+ NK cells. The flow cytometry measurements will be coupled with analysis 
of cell surface NK activating and inhibitory receptor expression. We will evaluate for 
maintenance of CD19-redirected effector function, using 51Cr release assay, CD107a 
degranulation,60,61 cytokine release (determined by intracellular cytokine assay for IFNγ and IL-


 
 
 
 
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2) and chemokine release (MIP1-α and MIP-1β), against CD19-expressing cell lines and, when 
available, primary CD19+ tumor cells collected and stored from recipients prior to treatment. 
 
Our preliminary data predict that we will be able to generate sufficient numbers of cells to meet 
the dose requirements of the proposed study,62 and that the CB-derived NK cells will have 
specificity against B-lymphoid malignancies (including ALL, CLL, NHL), without introducing 
unacceptable toxicity (Fig. 4-8). 
 
Even so, we are aware of several pitfalls that could compromise this projected outcome. 
Cytokine Release Syndrome. There have been recent reports of potentially life-threatening 
cytokine storm-related side-effects after adoptive therapy with CAR-transduced T cells, while 
prolonged B cell aplasia is a well-documented on-target toxicity of CAR19-transduced T 
cells.63;64  We will use the criteria for diagnosis and management defined in Appendices A. and 
B. to manage such patients. Moreover, to counteract these potential complications, we have 
incorporated a suicide gene based on the inducible caspase-9 gene into the CAR19 vector.54 
As shown in Fig 8, the addition of a small molecule dimerizer, AP1903, induces rapid apoptosis 
of transgenic cells, such that in the case of prolonged B lymphopenia, the dimerizer could be 
introduced to induce apoptosis of CAR19-transduced CB-NK cells, allowing normal recovery of 
B cells. This strategy would also be useful if the transduced NK cells are found to induce 
GVHD. 
 
3.0 Patient Eligibility 
Inclusion criteria: 
1. Patients with history of CD19 positive B-lymphoid malignancies, defined as acute 
lymphoblastic leukemia (ALL), diffuse large B-cell Lymphoma (DLBCL), chronic lymphocytic 
leukemia (CLL) small lymphocytic lymphoma (SLL), primary mediastinal large B-cell 
lymphoma, Richter’s transformation of CLL or SLL, follicular lymphoma, marginal zone 
lymphoma and high grade transformation of follicular or marginal zone lymphoma who have 
received at least 2 lines of standard chemoimmunotherapy or targeted therapy and have 
persistent disease. 
2. Patients with CD19 positive B-lymphoid malignancies as defined above with relapsed 
disease following standard therapy or a stem cell transplant. 
3. Patients at least 3 weeks from last cytotoxic chemotherapy at the time of starting 
lymphodepleting chemotherapy. Patients may continue tyrosine kinase inhibitors or other 
targeted therapies until at least three days prior to administration of lymphodepleting 
chemotherapy. 
4. Karnofsky/Lansky Performance Scale > 70. 
5. Adequate organ function: 
a. Renal: Serum creatinine </= 1.5 mg/dL or estimated Glomerular Filtration Rate (eGFR 
using the CKI-EPI equation) >/= 60 ml/min/1.73 m2. 
b. Hepatic: ALT/AST </= 2.5 x ULN or </= 5 x ULN if documented liver metastases, Total 
bilirubin </= 1.5 mg/dL, except in subjects with Gilbert’s Syndrome in whom total bilirubin 
must be </= 3.0 mg/dL. No history of liver cirrhosis. No ascites.  
c. Cardiac: Cardiac ejection fraction >/= 50%, no clinically significant pericardial effusion as 
determined by an ECHO or MUGA, and no clinically significant ECG findings. Clinical 
significance attributed per PI discretion. 
d. Pulmonary: No clinically significant, per PI discretion, pleural effusion, baseline oxygen 
saturation > 92% on room air. 
6. Able to provide written informed consent. 


 
 
 
 
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7. 7-80 years of age. 
8. All participants who are able to have children must practice effective birth control 
while on study and up to 3 months post completion of study therapy. Acceptable 
forms of birth control for female patients include: hormonal birth control, intrauterine 
device, diaphragm with spermicide, condom with spermicide, or abstinence, for the 
length of the study. If the participant is a female and becomes pregnant or suspects 
pregnancy, she must immediately notify her doctor. If the participant becomes 
pregnant during this study, she will be taken off this study. Men who are able to 
have children must use effective birth control while on the study. If the male 
participant fathers a child or suspects that he has fathered a child while on the 
study, he must immediately notify his doctor. 
9. Signed consent to long-term follow-up protocol PA17-0483. 
 
Exclusion Criteria: 
1. Positive beta HCG in female of child-bearing potential defined as not postmenopausal for 
24 months or no previous surgical sterilization or lactating females. 
2. Known positive serology for HIV. 
3. Presence of clinically significant Grade 3 or greater toxicity from the previous treatment, as 
determined by PI. 
4. Presence of fungal, bacterial, viral, or other infection requiring IV antimicrobials for 
management. Note: Simple UTI and uncomplicated bacterial pharyngitis are permitted if 
responding to active treatment. 
5. Presence of active neurological disorder(s). 
6. Concomitant use of other investigational agents. 
7. Previously received any anti-CD19 therapy (for example, CAR treatment or blinatumomab). 
 
4.0 Treatment Plan 
Lymphodepleting Chemotherapy (inpatient): 
 
On or before 
D-15 
Begin NK cell production 
 
D -6 
Admit / IV Hydration 
 
D -5 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m2 IV /  
                            Mesna 300 mg/m2 IV 
 
D -4 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m2 IV /  
                            Mesna 300 mg/m2 IV 
D -3 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m2 IV /                    
Mesna 300 mg/m2 IV 
D -2 
Rest 
D -1 
Rest 
D0 
Infusion of iC9/CAR.19/IL15-transduced CB-NK cells  


 
 
 
 
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Between D7  
& 14 
  Infusion of iC9/CAR.19/IL15-transduced CB-NK cells*  
 
Lymphodepleting Chemotherapy (outpatient): 
 
On or before 
D-15 
Begin NK cell production 
 
D -5 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m IV / 
Mesna 300 mg/m2 IV 
 
D -4 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m2 IV / 
Mesna 300 mg/m2 IV 
 
D -3 
Fludarabine 30 mg/m2 IV / Cyclophosphamide 300 mg/m2 IV / 
Mesna 300 mg/m2 IV 
D-2 
Rest 
 
D-1 
Rest 
 
D0 
Infusion of iC9/CAR.19/IL15-transduced CB-NK cells  
 
Between D7  
& 14 
Infusion of iC9/CAR.19/IL15-transduced CB-NK cells*  
 
* If no DLT is observed following the initial NK cell infusion, patients with bulky disease or 
rapidly progressive disease at study entry can receive at the investigators discretion  a 2nd 
NK cell infusion between days 7 and 14, using the same NK cell dose given initially, . 
 
 
Cell dose.  
Adult patients (i.e. 18 years or older)  who weigh 40 kilograms or more will receive a flat 
dose of  800 x106  ± 200x106 iC9/CAR.19/IL15-transduced CB-NK cells. Adult patients 
who weigh less than 40 kilograms and pediatric patients (younger than 18 years) will 
receive a dose of 107 iC9/CAR.19/IL15-transduced CB-NK cells per kilogram of weight.    
 
If the patient has relapsed or has persistent disease after a protocol assessment, an 
additional CAR NK infusions may be given. If there are left over cells from their first 
production, they may be used or a new cord unit may be selected for CAR NK 
generation. Prescreening testing will not need to be repeated if within 45 days of the 
previous tests, or at physician discretion. The lymphodepleting chemotherapy should be 
repeated if the additional infusion is given beyond 21 days of the date of completion of 
the last lymphodepleting chemotherapy. Patients with bulky or rapidly progressive 
disease can receive 2 consecutive infusions as described above 
 
Cyclophosphamide is dosed per adjusted body weight for patients weighing > 20% 


 
 
 
 
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above their ideal body weight. For patients less than or equal to 20% above their ideal 
body weight, the actual body weight is used. 
 
On D 0 the NK cell infusion will be administered intravenously. Premedicate with 
Benadryl 25 mg po or IV and Tylenol 650 mg po. The use of steroids is contraindicated 
unless required for physiologic replacement. 
 
Subjects may be either inpatient or outpatient for the CAR NK infusion, depending upon 
bed availability and/or patient's clinical situation. Vital signs (temperature, heart rate, 
blood pressure, and respiratory rate) will be obtained on all patients per BMT standard 
of care for cellular therapy which includes at the start of the CAR NK infusion and 
approximately every 15 minutes x 4 followed by approximately every 30 minutes x 2 or 
until 1 hour after completion of the CAR NK infusion.  Then approximately every hour as 
indicated by patient’s condition. 
 
NK cells will be obtained by the following method: 
Frozen cord blood units will be thawed and mononuclear cells will be isolated by 
Ficoll density gradient centrifugation. NK cells will be CAR transduced and generated 
for 14 to 22 days in liquid cultures using APC feeder cells as described in detail in the 
Chemistry, Manufacturing and Controls (CMC). 
 
NK Product Release Criteria 
The following minimum criteria will be required for release of the expanded NK cells for 
reinfusion: 
 
Stat Gram Stain: “No Organisms 
Seen”. CAR+ NK cells: > 15% 
CD3+ number: < 2 e5 CD3+ 
cells/kg. CD32+ cell number 
(aAPV): < 5% 
NK cells (CD16+/56+): >80% 
Visual Inspection: “No Evidence of Contamination” (turbidity; change in media color). 
Endotoxin Assay: < 5EU/Kg. 
Viability: ≥ 70%. 
 
Other parameters which will be monitored include sterility culture for bacteria and fungi. 
If more than 2 x 10e5 CD3+ cells/kg are present, a second cycle of CD3 depletion 
may be performed. The cell dose for infusion may be reduced so that the infused 
CD3+ cells are <2 x10 5/ kg. 
 
If adequate CAR+ NK cell dose is not generated, then all available cells will be infused. If 
this occurs for patients in the MTD finding stage of this study, they will not be counted in 
any cohort. If more than the required NK dose is generated, the additional NK cells may 
be cryopreserved for future infusions or may be used for research. 
 
If CAR NK cells cannot be released due to microbial contamination, another cord blood 
unit will be selected and production will start over. If the patient has already completed 
lymphodepleting chemotherapy, they may require a second dose of lymphodepleting 
chemotherapy prior to the CAR NK infusion. 
 


 
 
 
 
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Administration of the Dimerizer AP1903 for Cytokine Release Syndrome (CRS), 
Neurotoxicity, or GVHD. 
 
The MD Anderson CARTOX committee has developed the detailed algorithms for the 
management of CRS or neurotoxicity summarized in Appendix A. We will follow the 
algorithm rigorously with the administration of Tocilizumab 8 mg/kg IV q 6h as needed for 
up to 3 doses / 24h for Grade 2 CRS or Grade 2 Neurotoxicity not responding to standard 
supportive measures. For Grade 3 CRS and Grade 3 Neurotoxicity as defined in the 
CARTOX algorithm, in addition to the Tocilizumab, a single dose of AP1903 will be 
administered (0.4 mg/kg as an intravenous infusion over approximately 2 hours). The 
AP1903 dose is based on published Pk data which show plasma concentrations of 10-
1275 ng/mL over the 0.01 mg/kg to 1.0 mg/kg dose range with plasma levels falling to 18% 
and 7% of maximum at 0.5 and 2hrs post dose.65  The dimerizer can also be used for the 
treatment of grades I-IV GVHD. Responses in patients with GVHD who had received 
Capsase-9+ T cells and then the AP1903, responses have occurred within the first 24-48 
hours. 
 
Patients who do not experience downgrading or CRS or neurotoxicity to Grade 2 or less 
within 12 hours may receive a second dose of AP1903 but will also receive high dose 
steroids as outlined in the CARTOX Algorithm in Appendix A. and Appendix B. 
 
Birth Control Specifications 
 
All participants who are able to have children must practice effective birth control while on 
study and up to 3 months post completion of therapy. Acceptable forms of birth control for 
female patients include: hormonal birth control, intrauterine device, diaphragm with 
spermicide, condom with spermicide, or abstinence, for the length of the study. If the 
participant is a female and becomes pregnant or suspects pregnancy, she must 
immediately notify her doctor. If the participant becomes pregnant during this study, she 
will be taken off this study. Men who are able to have children must use effective birth 
control while on the study. If the male participant fathers a child or suspects that he has 
fathered a child while on the study, he must immediately notify his doctor. 
 
Additional Therapies 
 
If the patient has persistent disease after a protocol assessment, standard of care 
therapies may be given at discretion of the physician and PI. The patient will remain on 
study to monitor for persistence of CAR NK cells and may be considered for an additional 
infusion. 
5.0 Evaluation During Study 
Evaluation During Study 
Every effort will be made to adhere to the schedule of events and all protocol 
requirements. Variations in schedule of events and other protocol requirements that do 
not affect the rights and safety of the patient will not be considered as deviations. Such 
variations may include laboratory assessments completed outside of schedule and 
occasional missed required research samples. Missed samples for correlative studies 


 
 
 
 
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will not constitute protocol deviations. 
 
5.1 
Evaluation: Any time before or during study: HLA typing (high resolution A,B, DR). 
5.2 
The following evaluations will be obtained within 30 days of study enrollment: 
 
5.2.1 
History and physical examination 
5.2.2 
CBC w/diff and platelets, total bilirubin, SGPT, alkaline phosphatase, 
LDH, albumin, total protein, BUN, creatinine, glucose, electrolytes, 
PT/PTT, type and screen, immunoglobulin levels (IGG, IGM, IGA), and 
cytokine panel 3 (IL6, IFN gamma, TNF alpha). 
5.2.3 
Serology for HIV. 
5.2.4 
ECHO or MUGA. 
5.2.5 
Pulmonary function tests, if clinically indicated. 
5.2.6 
Chest x-ray 
5.2.7 
Urinalysis. 
5.2.8 
CT brain. 
5.2.9 
PET/CT scan as clinically indicated. 
5.2.10 Bone marrow aspiration as clinically indicated. 
5.2.11 EKG 
 
5.3 
The following blood will be drawn after the consent is signed but 
before the lymphodepleting chemotherapy is initiated: 
 
5.3.1 
Research Labs, consisting of 60 mLs of blood (5 x green top and 1 
purple top) will be drawn and sent to Dr. Rezvani’s laboratory. The 
studies will be performed free of charge to the patient. 
 
5.4 
Evaluations within 7 days of starting lymphodepleting chemotherapy: 
 
5.4.1 
History and physical examination including weight and vital signs. 
5.4.2 
Laboratory examinations: CBC w/diff and platelets, total bilirubin, SGPT, 
alkaline phosphatase, LDH, albumin, total protein, BUN, creatinine, 
glucose, electrolytes, and cytokine analysis. Serum pregnancy test if 
female participant of childbearing potential. 
 
5.5 
The following evaluations will be obtained on approximately: day 0, day 3, 
day 7, day 14, and day 21, week 4, week 8, week 12, week 16, month 6, 
month 9, and 1 year after CAR-NK infusion: 
 
5.5.1 
Physical examination including weight and vital signs at Day 7 only. 
5.5.2 
CBC w/diff and platelets, chemistry panel, and cytokine analysis. 
5.5.3 
Cytokine panel 3 (IL6, IFN gamma, TNF alpha) at all time points, except 
only as clinically indicated at approximately month 6, month 9, and 
month 12. 
5.5.4 
HLA antibodies at approximately 4 weeks and 12 weeks only. 
5.5.5 
Research Labs: CAR NK detection, phenotype and function to be 
performed in Dr. Katayoun Rezvani’s Laboratory as above. 
 
 
 


 
 
 
 
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5.6 
The following evaluations will be obtained on approximately week 4, 
week 8, week 12, week 16, and month 6, month 9, and month 12 after CAR-
NK infusion: 
 
5.6.1 
PET/CT scan as clinically indicated. 
 
5.7 
The following evaluations will be obtained on approximately day 7, week 4, week 
 
8, week 12, week 16, month 6, month 9, and 1 year after CAR-NK infusion: 
 
5.7.1 
Bone marrow aspiration and/or biopsy as clinically indicated. 
5.7.2 
Research Labs: 5 to 10 mL bone marrow aspirate will be sent to Dr. 
Rezvani’s Laboratory, as clinically indicated. 
 
5.8 
Additional lab tests 
 
Additional lab draws may be needed to monitor the car NK cells and patient 
disease status. The patient may be asked to sign a separate consent for protocol 
LAB00-099 for these additional samples. Analysis will be performed in Dr. Katy 
Rezvani's Lab free of charge for the patient. Analysis results from these samples 
performed under protocol LAB00-099 will be used in protocol 2016-0641. 
 
5.9 
Lymph node biopsy 
 
If the patient has a diagnostic lymph node biopsy, a portion of the specimen may 
be sent to Dr. Katy Rezvani's Lab for analysis, if available. 
 
 


 
 
 
 
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Table 1- Table of Evaluations 
Pre Day 0 
3 
days 
7 
days 
14 
days 
21 
days 
4 
weeks 
Day 
40 
8 
weeks 
12 
weeks 
16 
weeks 
6, 9, 12 
months 
Physical Exam 
x1 
 
x1 
CBC, chem panel 
 
x1 
x 
x 
x 
x 
x 
x 
x 
x 
x2 
HIV serology 
x 
 
Cytokine Panel3 
 
x4 
 
x 
x 
x 
x 
x 
x 
x 
x2 
HLA antibodies 
x 
x 
Research labs: CAR NK 
detection, phenotype and 
function 
x 
x6 
x 
x 
x 
x 
x 
x 
x 
x 
x2 
3 
Cytokine Analysis 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x2 
3 
Chimerism 
x 
x 
x 
x 
x 
x 
x 
x 
x2 
5 
RCR blood test 
x 
x 
x2 
Pregnancy Test2 
x1 
Urinalysis 
x 
EKG 
x 
ECHO or MUGA 
x 
Pulmonary Function Test x2 
Brain CT 
x 
PET/CT 
 
x 
 
x2 
x2 
x2 
x2 
x2 
Bone marrow analysis 
 
x2 
x2 
x2 
 
x2 
x2 
x2 
x2 
AE assessment 
< ================================================ > 
Bone marrow research 
labs to assess NK CAR 
homing 
x 
x 
x2 
x2 
x2 
x2 
x2 
 
Time frame windows: Protocol assessments will be done approximately on days listed above. For 
patients who receive a second CAR NK infusion, the post testing timepoints will be based from date of 
second infusion. 
 
1  Physical Exam, CBC, chem panel: Within 30 days and 7 days of starting lymphodepleting 
chemotherapy. Pregnancy test: Within 7 days of starting lymphodepleting chemotherapy. 
Physical at approximately Day 7 only. 
2  As clinically indicated. 
3  Drawn as part of the research lab z code. Samples will be batched and run approximately 
every 3 months for results. 
4 Cytokine Panel 3: Day 0 prior to CAR NK infusion. 
5 Replication-competent retrovirus (RCR): About 1, 3, 6 and 12 months post NK cell infusion, 
and then per long term follow up study PA17-0483. 
6 On day 0 a research sample will be drawn before infusion, may be with morning labs, and then 
approximately 1-3 hours after completion. 
 
 


 
 
 
 
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6.0 Background Drug Information 
The medications used in this protocol will be obtained through a commercially available 
supply. 
 
Cyclophosphamide: 
 
Therapeutic Classification: Antineoplastic alkylating agent 
 
Pharmaceutical Data: Lyophilized cyclophosphamide for injection contains 75 mg of 
mannitol per 100 mg of cyclophosphamide (anhydrous) and is supplied in vials for 
single dose use. 
 
Stability and Storage: Storage at or below 77°F (25°C) is recommended; this product 
will withstand brief exposure to temperatures up to 86°F(30°C) but should be 
protected from temperatures above 86°F. Reconstituted lyophilized cyclophosphamide 
for injection is chemically and physically stable for 24 hours at room temperature or for 
6 days in the refrigerator; it does not contain any antimicrobial preservative and thus 
care must be taken to assure the sterility of prepared solutions. 
 
Mechanism of Action: It interferes with DNA replication and transcription of RNA 
and ultimately results in the disruption of nucleic acid function. 
 
Side effects: Dose limiting toxicity is hematologic. Nausea and vomiting are common. 
Anorexia, less frequently abdominal discomfort or pain and diarrhea may occur. There 
are isolated reports of hemorrhagic colitis, oral mucosal ulceration and jaundice 
occurring during therapy. Azotemia has been reported. Alopecia and hypersensitivity 
reactions can occur when high doses are utilized. Hemorrhagic cystitis may develop in 
patients treated with cyclophosphamide. Rarely this condition can be severe and even 
fatal. Acute cardiotoxicity has been reported with doses as low as 2.4 g/m Interstitial 
pulmonary fibrosis has been reported in patients receiving high doses of 
cyclophosphamide over a prolonged period. 
 
Fludarabine: 
 
Therapeutic Classification: Antineoplastic agent; Antimetabolite (Purine Analog) 
 
Pharmaceutical Data: Each vial contains 50 mg lyophilized drug, to be reconstituted 
with 2 ml sterile water to a solution that is 25 mg/ml for IV administration. 
 
Solution Preparation: Mix each vial with 2 ml sterile pyrogen-free water to a clear 
solution, which is 25 mg/ml for IV administration only. Reconstituted solution should be 
used within 8 hours. 
 
Adverse Effects: 
Observed in more than 10%: Cardiovascular: Edema (8% to 19%); Central nervous 
system: Fever (11% to 69%), fatigue (10% to 38%), pain (5% to 22%), chills (11% to 
19%); Dermatologic: Rash (4% to 15%); Gastrointestinal: Nausea/vomiting (1% to 
36%), anorexia (34%), diarrhea (5% to 15%), gastrointestinal bleeding (3% to 13%); 
Genitourinary: Urinary tract infection (2% to 15%); Hematologic: Myelosuppression 


 
 
 
 
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(nadir: 10-14 days; recovery: 5-7 weeks; dose-limiting toxicity), anemia (14% to 60%), 
neutropenia (grade 4: 37% to 59%; nadir: ~13 days), thrombocytopenia (17% to 55%; 
nadir: ~16 days); Neuromuscular & skeletal: Weakness (9% to 65%), myalgia (4% to 
16%), paresthesia (4% to 12%); Ocular: Visual disturbance (3% to 15%); Respiratory: 
Cough (£44%), pneumonia (3% to 22%), dyspnea (1% to 22%), upper respiratory 
infection (2% to 16%), rhinitis (£11%); Miscellaneous: Infection (12% to 44%), 
diaphoresis (£14%) 
 
Observed in 1% to 10%: Cardiovascular: Peripheral edema (less than 7%), angina (less 
than 6%), chest pain (less than 5%), CHF (3%), arrhythmia (3%), cerebrovascular 
accident (3%), MI (3%), supraventricular tachycardia (3%), deep vein thrombosis (1% to 
3%), phlebitis (1% to 3%), aneurysm (less than 1%), transient ischemic attack (1%); 
Central nervous system: Headache (9%), malaise (6% to 8%), sleep disorder (1% to 3%), 
cerebellar syndrome (less than 1%), depression (1%), mentation impaired (1%); 
Dermatologic: Alopecia (3%), pruritus (1% to 3%); Endocrine & metabolic: Hyperglycemia 
(1% to 6%), LDH increased (less than 6%), dehydration (1%); Gastrointestinal: Abdominal 
pain (10%), stomatitis (9%), weight loss (6%), esophagitis (3%), constipation (1% to 3%), 
mucositis (2%), dysphagia (1%); Genitourinary: Dysuria (3% to 4%), hesitancy (3%); 
Hematologic: Hemorrhage (1%), myelodysplastic  lyndrome/acute myeloid leukemia 
(usually associated with prior or concurrent treatment with other anticancer agents); 
Hepatic: Cholelithiasis (3%), liver function tests abnormal (1% to 3%), liver failure (1%); 
Neuromuscular & skeletal: Back pain (9%), osteoporosis (2%), arthralgia (1%); Otic: 
Hearing loss (2% to 6%); Renal: Hematuria (2% to 3%), renal failure (1%), renal function 
test abnormal (1%), proteinuria (1%); Respiratory: Bronchitis (9%), pharyngitis (9%), 
allergic pneumonitis (6%), hemoptysis (1% to 6%), sinusitis (5%), epistaxis (1%), hypoxia 
(1%); Miscellaneous: Flu-like syndrome (5% to 8%), herpes simplex infection (8%), 
anaphylaxis (1%), tumor lysis syndrome (1%) 
 
Observed in <1%, postmarketing, and/or case reports: Acute respiratory distress 
syndrome, agitation, blindness, blurred vision, bone marrow fibrosis, coma, confusion, 
diplopia, eosinophilia, Epstein-Barr virus (EBV) associated lymphoproliferation, EBV 
reactivation, erythema multiforme, Evans syndrome, flank pain, hemolytic anemia 
(autoimmune), hemophilia (acquired), hemorrhagic cystitis, herpes zoster reactivation, 
hyperkalemia, hyperphosphatemia, hyperuricemia, hypocalcemia, interstitial 
pneumonitis, metabolic acidosis, opportunistic infection, optic neuritis, optic neuropathy, 
pancreatic enzymes abnormal, pancytopenia, pemphigus, pericardial effusion, peripheral 
neuropathy, photophobia (primarily with high doses), progressive multifocal 
leukoencephalopathy (PML), pulmonary fibrosis, pulmonary hemorrhage, pulmonary 
infiltrate, respiratory distress, respiratory failure, Richter’s syndrome, seizure, skin cancer 
(new onset or exacerbation), Stevens-Johnson syndrome, thrombocytopenia 
(autoimmune), thrombocytopenic purpura (autoimmune), toxic epidermal necrolysis, 
trilineage bone marrow aplasia, trilineage bone marrow hypoplasia, urate crystalluria, 
wrist drop 
 
Also observed: Neurologic syndrome characterized by cortical blindness, coma, and 
paralysis [36% at doses >96 mg/m2 for 5-7 days; <0.2% at doses <125 mg/m2 /cycle 
(onset of neurologic symptoms may be delayed for 3-4 weeks)] 
 
Mechanism of Action: Fludarabine inhibits DNA synthesis by inhibition of DNA polymerase 
and ribonucleotide reductase; also inhibits DNA primase and DNA ligase I. 


 
 
 
 
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Human Safety and Pharmacology. 
 
Distribution: Vd: 38-96 L/m2; widely with extensive tissue binding 
Protein binding: 2-fluoro-ara-A: ~19% to 29% 
Metabolism: I.V.: Fludarabine phosphate is rapidly dephosphorylated in the plasma to 
2-fluoro-ara-A (active metabolite), which subsequently enters tumor cells and is 
phosphorylated by deoxycytidine kinase to the active triphosphate derivative 
(2-fluoro-ara-ATP) 
Bioavailability: Oral: 2-fluoro-ara-A: 50% to 65% Half-life elimination: 2-fluoro-ara-A: 
~20 hours Time to peak, plasma: Oral: 1-2 hours 
Excretion: Urine (60%, 23% as 2-fluoro-ara-A) within 24 hours 
 
Dose adjustment in renal and hepatic impairment: It appears that no adjustment is 
needed in hepatic impairment. Renal impairment dosing is NOT specific to stem cell 
transplant patients. In patients not receiving a stem cell transplant, doses are typically 
reduced by 20% for CrCl of 30-70 ml/min and not used for CrCl < 30 ml/min. 
 
Monitoring Parameters: CBC with differential, platelet count, AST, ALT, serum creatinine, 
serum albumin, uric acid; monitor for signs of infection and neurotoxicity 
 
Mesna (sodium -2-mercapto ethane sulphonate) 
 
Mesna is a prophylactic agent used to prevent hemorrhagic cystitis induced by the 
oxazophosphorines (cyclophosphamide and ifosfamide). It has no intrinsic cytotoxicity 
and no antagonistic effects on chemotherapy. Mesna binds with acrolein, the urotoxic 
metabolite produced by the oxazophosphorines, to produce a non-toxic thioether and 
slows the rate of acrolein formation by combining with 4-hydroxy metabolites of 
oxazophosphorines. At the doses used for uroprotection, mesna is virtually non-toxic. 
However, adverse effects which may be attributable to mesna include nausea and 
vomiting, diarrhea, abdominal pain, altered taste, rash, urticaria, headache, joint or limb 
pain, hypotension and fatigue. 
 
7.0 Criteria for Removal from the Study 
1. Patient withdrawal of the informed consent/authorization, or refusal to continue on study. 
2. An increasing or unexpected pattern of toxicity is observed deemed unacceptable by 
the PI, co-PI, or designee. 
3. Inability to infuse CAR-NK cell product due to product manufacturing issues. 
4. Study completion at one year. 
5. Death. 
6. Not able to receive CAR NK infusion(s). 
7. Non-compliance with study procedures and monitoring. 
8. Lost to follow-up. 
9. Disease progression, 
10. If for any reason, the PI feels it in the best interest of the patient to come off study. 
 
 
 


 
 
 
 
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8.0 Statistical Considerations 
1. Preliminaries This is a single-arm phase II trial of cord blood derived CAR CD-19 natural 
killer (NK) cells, administered at a dose of 800x10e6 cells to patients with any hematologic 
malignancy who have either relapsed or have been declared refractory after initial 
chemotherapy. The primary scientific goal of the trial is to estimate the probabilities of 
treatment efficacy and toxicity. 
 
2. Outcomes.  
Toxicity is defined as grade 3 or 4 GVHD within 40 days of NK cell infusion or cytokine 
release syndrome (CRS) within 2 weeks of NK cell infusion requiring transfer to intensive 
care. 
Efficacy is defined as the patient being alive and in at least partial remission at day 30 post 
NK cell infusion. 
Secondary outcomes. Secondary outcomes will include progression-free survival (PFS) 
time, overall survival (OS) time, and response at day 100. 
3.  Maximum Sample Size and Cohort Size. A maximum of 120 patients will be treated in 
up to 8 cohorts of size 15 patients each.   This sample size ensures that, if for example 
36/120 (30%) of the patients have the Efficacy outcome, then starting with a beta(.30, .70) 
prior, a posterior 95% credible interval for Pr(Efficacy) would be .22 -  .38 .  Similarly, if 
48/120 patients experience toxicity, then starting with a beta(.40, .60) prior, a posterior 95% 
credible interval for Pr(Toxicity) would be .31 - .49. 
  
4. Safety and Futility Monitoring.   The method of Thall et al69 will be used to construct two 
early stopping bounds, one for inadequate efficacy and one for excessive toxicity, that will be 
applied simultaneously. For the purpose of monitoring Efficacy and Toxicity, a maximum 
upper limit on Pr(Toxicity) will be .40, and a minimum lower limit on Pr(Efficacy) will be .30.  
Note that, because Efficacy includes 30-day survival as a subevent, monitoring the probability 
of Efficacy includes monitoring of early death. 
 
Assuming that Efficacy and Toxicity and independent events, the four composite 
elementary events will be denoted by A1 = [Efficacy and Toxicity], A2 =  [Efficacy and No 
Toxicity],  A3 = [No Efficacy and Toxicity], and  A4 =  [No Efficacy and No Toxicity].  Thus, 
Efficacy = A1 U A2 and Toxicity = A1 U A3. Denoting the respective probabilities of these 
4 elementary events with the experimental treatment E = cord blood derived CAR CD-19 
natural killer (NK) cells, by q =  (q1, q2, q3, q4), so that q1 + q2 + q3 +q4 = 1, As a 
standard, S, for comparison, the historical treatment distribution for use as a comparator, 
with probabilities p =  (p1, p2, p3, p4). With E, the probability of Efficacy be denoted by qE 
= q1 + q2, and the probability of Toxicity will be denoted by  qT = q1 + q3. With S, the 
probability of Efficacy be denoted by pE = p1 + p2, and the probability of Toxicity will be 
denoted by  pT = p1 + p3. It will be assumed, conservatively, that q follows a Dirichlet prior 
distribution with parameters (.12, .18,  .28, .42), implying that qE follows a beta prior with 
parameters (.30, .70). It will be assumed that p follows a Dirichlet distribution with 
parameters (120, 180,  280, 420).    
 
The trial will be stopped early for lack of Efficacy if Pr(pE < qE | data)   < .01 


 
 
 
 
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The trial will be stopped early for unacceptable Toxicity if Pr(pT < qT | data)   > .99 
 
These posterior probability criteria imply that the trial will be stopped early if either 
 
[number of patients with efficacy] / [number of patients evaluated] is less than or equal to 
 
0/15, 3/30, 6/45, 9/60, 13/75, 16/90, or 20/105,  
 
or 
 
[number of patients with toxicity] / [number of patients evaluated] is greater than or equal 
to 
 
11/15, 19/30, 27/45, 34/60, 41/75, 48/90, or 55/105. 
 
5. Operating Characteristics 
 
The design’s operating characteristics are summarized in Table 1. The two monitoring 
rules will be applied after successive cohorts of 15 without halting the enrolment while 
the data is being analyzed. The simulations were carried out using multc99. 
 
Table 1.  Design operating characteristics, based on Nmax = 120, with the two monitoring 
rules applied after successive cohorts of 15, at interim sample sizes of 15, 30, 45, 60, 75, 
90 and 105.  
 
Scenario 
p1 
p2 
p3 
p4 
Pr(Eff) 
Pr(Tox) 
Pr(Stop 
Early) 
Sample Size 
25th, 50th, 75th 
percentiles 
1 
.12 
.18 
.28 
 
 .42 
.30 
.40 
0.06 
120  120  120 
2 
.05 
.05 
.35 
.55 
.10 
.40 
1.00 
30  30  45   
3 
.12 
.18 
.48 
.22 
.30 
.60 
0.96 
30  45  60 
4 
.05 
.05 
.55 
.35 
.10  
.60 
1.00 
15  30  30 
 
In scenario 1, both Pr(Eff) and Pr(Tox) are acceptable. 
 
In scenario 2,  Pr(Eff) = .10 is too low.    
 
In scenario 3, Pr(Tox) = .60 is too high. 
 
In scenario 4, both Pr(Eff) and Pr(Tox) are unacceptable. 
 
6. Data analyses 
Unadjusted distributions of the time-to-event outcomes OS and PFS will be estimated using the 
method of Kaplan and Meier.70 Their relationship to prognostic covariates and OS or pFS will be 
evaluated by Bayesian piecewise exponential survival regression.71  
 
 
 


 
 
 
 
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7.  Criteria for Response 
 
For the purpose of the Efftox assessment, a response is defined as a partial or complete 
response. 
· 
Acute lymphoblastic leukemia: Complete response will be defined as bone marrow 
with < 5% blasts, the absence of circulating blasts, and no extramedullary sites of 
disease (as assessed by means of computed tomography or positron-emission 
tomography), regardless of cell-count recovery. 
· 
CLL: Response will be defined based on the NCI-WG/IWCLL 2008 criteria, 
Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report 
from the International Workshop on Chronic Lymphocytic Leukemia updating the 
National Cancer 
Institute–Working Group 1996 guidelines.72   
Lymphomas: Response will be defined based on the Lugano criteria- Recommendations for 
Initial Evaluation, Staging, and Response Assessment of Hodgkin and Non-Hodgkin 
Lymphoma:  The Lugano Classification.73   
 
The Investigator is responsible for completing toxicity summary reports and submitting 
them to the IND office Medical Monitor for review.  These should be submitted as follows:  
 
After the first 15 evaluable patients, complete 40 days of study treatment, and every 15 
evaluable patients thereafter. 
 
9.0 Adverse Events and Reporting Requirements 
An Adverse Event is defined as any untoward medical occurrence in a patient 
regardless of its causal relationship to study treatment. An AE can be any unfavorable 
and unintended sign (including any clinically significant abnormal laboratory test 
result), symptom, or disease temporally associated with the use of the study 
treatment, whether or not it is considered to be study drug(s) related. Included in this 
definition are any newly occurring events and any previous condition that has 
increased in severity or frequency since the administration of study therapy. 
Attribution - the determination of whether an adverse event is related to a medical 
treatment or procedure.  
• 
Definite - the adverse event is clearly related to the investigational agent(s). 
• 
Probable - the adverse event is likely related to the investigational agent(s). 
• 
Possible - the adverse event may be related to the investigational agent(s). 
• 
Unlikely - The adverse event is doubtfully related to the investigational agent(s). 
• 
Unrelated - The adverse event is clearly NOT related to the investigational agent(s).  
For the purpose of this study, the investigational component of the treatment plan is the 
infusion of ex vivo-expanded, genetically modified, iC9/CAR.19/IL15-transduced NK 
cells. Therefore, serious and unexpected adverse events occurring from the infusion of 
the iC9/CAR.19/IL15-transduced NK cells and up to 30 days of the last treatment 


 
 
 
 
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intervention, as defined below, will be reported according to MDACC policy and 
procedures below and the (OBA) Serious Adverse Event Reporting Form for Human 
Gene Transfer Clinical Studies at http://www4.od.nih.gov/oba/rac/documents1.htm. 
 
The MDACC Internal Adverse Event Reporting Form will be used for reporting to the IRB 
and the Investigational New Drug (IND) Office. Serious adverse events must be followed 
until clinical recovery and laboratory tests have returned to baseline, progression of the 
event has stabilized, or there has been acceptable resolution of the event. Additionally, 
any serious adverse events occurring after the 30 day time period that are related to the 
study treatment must be reported to the IRB and the IND Office. This includes the 
development of a secondary malignancy. 
 
Hospitalizations for treatment of disease, or related to complications from treatment 
administered prior to CAR infusion will not be captured. 
 
Collection of adverse events (AEs) will begin at the time of the iC9/CAR.19/IL15-
transduced NK cell infusion and will reflect the onset and resolution date and maximum 
grade. BMTWeb/CORe will be used as the electronic case report form for this protocol, 
and all protocol specific data will be entered into BMTWeb/CORe. All protocol specific 
data defined as abnormal physical exam and/or laboratories and AEs related to 
iC9/CAR.19/IL15-transduced NK cell infusion will be entered into BMTWeb/CORe. 
Intermittent events should be labeled as such and followed until resolution. If a patient is 
taken off study while an event is still ongoing, this will be followed until resolution unless 
another therapy is initiated. Additionally, patients may be on multiple concurrent 
medications that are not necessarily related to the iC9/CAR.19/IL15-transduced NK cell 
infusion. These medications are considered standard of care and have no scientific 
contributions to the protocol, therefore concurrent medications will not be captured. 
 
MD Anderson's Clinical Research Serious Adverse Events Reporting (eSAE) system will 
be used to collect and report all serious adverse event information. 
 
Assessment of adverse events 
The Investigator or physician designee is responsible for verifying and providing source 
documentation for all adverse events and assigning the attribution for each event for all 
subjects enrolled on the trial. 
 
Adverse events related to the iC9/CAR.19/IL15-transduced NK cell infusion and protocol 
specific data will be entered into PDMS/CORe. PDMS/CORe will be used as the 
electronic case report form for this protocol. 
 
Severity of the adverse events (AEs) 
All grades of AEs related to iC9/CAR.19/IL15-transduced NK cell infusion will be 
collected. All AEs will be recorded on the AE log. Adverse events unrelated to the 
iC9/CAR.19/IL15-transduced NK cell infusion will not be collected. 
 
Events not included in the CTCAE chart will be scored as follows: 
 
General grading: 
Grade 1: Mild: discomfort present with no disruption of daily activity, no treatment 


 
 
 
 
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required beyond prophylaxis. 
Grade 2: Moderate: discomfort present with some disruption of daily activity, require 
treatment. Grade 3: Severe: discomfort that interrupts normal daily activity, not 
responding to first line treatment. 
Grade 4: Life Threatening: discomfort that represents immediate risk of death 
 
Causality Assessment 
The investigational component of the treatment plan of this study is the infusion of ex-
vivo expanded iC9/CAR.19/IL15-transduced NK cells after lymphodepleting 
chemotherapy. 
 
Therefore, events known to be caused by the iC9/CAR.19/IL15-transduced NK cell 
infusion and its direct consequences will be assessed as definitely related when 
assessing the causality. 
When the relationship of the adverse event cannot be rule out between the 
iC9/CAR.19/IL15-transduced NK cell infusion and the chemotherapy, the event will be 
scored as probably or possible related. 
 
Events known to be related to drugs used as chemotherapy as well as to drugs used as 
supportive treatment will be scored as unrelated to the iC9/CAR.19/IL15-transduced NK 
cell infusion. 
 
The principal investigator will be the final arbiter in determining the causality assessment. 
 
AEs related to the iC9/CAR.19/IL15-transduced NK cell infusion: 
1. These expected events will be monitored at approximately 30 minutes and 60 
minutes post infusion: 
· 
Fever, 
· 
Chills, 
· 
Decrease in blood pressure, 
· 
Rash, 
· 
Shortness of breath. 
 
2. Dose limiting, (also considered unexpected): 
· 
Grade 4 NK infusion related toxicity, 
· 
Grades 3-5 allergic reactions related to study cell infusion, 
· 
Grade 3-5 organ toxicity (cardiac, dermatologic, gastrointestinal, hepatic, 
pulmonary, renal/genitourinary, or neurologic) not pre-existing or due to the 
underlying malignancy or due to lymphodepleting chemotherapy 
· 
Grades 3-4 acute GVHD occurring within 8 weeks. 
· 
Treatment-related death within 8 weeks of the study cell infusion. 
Adverse Events Considered Serious: 
· 
Grade 4 NK infusion related toxicity. 
· 
GvHD. 
· 
Prolonged hospitalization due to infections and/or organ failure requiring 
extensive supportive care (i.e. dialysis, mechanical ventilation).    
· 
Any expected or unexpected event resulting in an irreversible condition and/ or 
leading to death. 
 


 
 
 
 
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Abnormal Laboratory Findings 
 
For the purpose of this study, abnormal laboratory findings considered associated to the 
original disease as well as isolated changes in laboratory parameters such as electrolyte 
magnesium and metabolic imbalances, uric acid changes, elevations of GPT, GOT, LDH, 
alkaline phosphatase, and CBC would not be considered adverse events and will not be 
collected in the database or toxicity logs. 
 
Concomitant Medications 
 
Patients treated on this protocol will require supportive care treatment (concomitant 
medications). These medications are considered standard of care and have no scientific 
contribution to the protocol; therefore no data will be captured on various medications 
needed or their side effects. 
 
Serious Adverse Event Reporting (SAE) 
 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of 
either the investigator or the sponsor, it results in any of the following outcomes: 
 
· 
Death 
· 
A life-threatening adverse drug experience – any adverse experience that places 
the patient, in the view of the initial reporter, at immediate risk of death from the 
adverse experience as it occurred. It does not include an adverse experience that, 
had it occurred in a more severe form, might have caused death. 
· 
Inpatient hospitalization or prolongation of existing hospitalization 
· 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 
· 
A congenital anomaly/birth defect. 
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. 
Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do not 
result in inpatient hospitalization, or the development of drug dependency or drug abuse 
(21 CFR 312.32). 
 
· 
Important medical events as defined above, may also be considered serious 
adverse events. Any important medical event can and should be reported as an 
SAE if deemed appropriate by the Principal Investigator or the IND Sponsor, 
IND Office. 
 
· 
All events occurring during the conduct of a protocol and meeting the definition of 
a SAE must be reported to the IRB in accordance with the timeframes and 
procedures outlined in “The University of Texas M. D. Anderson Cancer Center 
Institutional Review Board Policy for Investigators on Reporting Unanticipated 
Adverse Events for Drugs and Devices”. 


 
 
 
 
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Unless stated otherwise in the protocol, all SAEs, expected or unexpected, must be 
reported to the IND Office, regardless of attribution (within 5 working days of 
knowledge of the event). 
· 
All life-threatening or fatal events, that are unexpected, and related to the study 
drug, must have a written report submitted within 24 hours (next working day) of 
knowledge of the event to the Safety Project Manager in the IND Office. 
 
· 
Unless otherwise noted, the electronic SAE application (eSAE) will be 
utilized for safety reporting to the IND Office and MDACC IRB. 
 
· 
Serious adverse events will be captured from the time of the first protocol-
specific intervention, until 30 days after the last dose of drug, unless the 
participant withdraws consent. Serious adverse events must be followed until 
clinical recovery is complete and laboratory tests have returned to baseline, 
progression of the event has stabilized, or there has been acceptable 
resolution of the event. 
 
· 
Additionally, any serious adverse events that occur after the 30 day time 
period that are related to the study treatment must be reported to the IND 
Office. This may include the development of a secondary malignancy. 
 
· 
The gene therapy reporting addendum (“Additional Reporting Form for Serious 
Adverse Events on Gene Transfer Trials”) must be included with each SAE 
submitted. 
 
Reporting to FDA: 
 
· 
Serious adverse events will be forwarded to FDA by the IND Sponsor (Safety 
Project Manager IND Office) according to 21 CFR 312.32. 
 
It is the responsibility of the PI and the research team to ensure serious adverse 
events are reported according to the Code of Federal Regulations, Good Clinical 
Practices, the protocol guidelines, the sponsor’s guidelines, and Institutional 
Review Board policy. 
 
 
 
 


 
 
 
 
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10.0 
References 
 
1. 
Cheson BD, Cassileth PA, Head DR et al. Report of the National Cancer 
Institute-sponsored workshop on definitions of diagnosis and response in acute 
myeloid leukemia. J.Clin.Oncol. 1990;8:813-819. 
 
2. 
Burger JA, Tedeschi A, Barr PM et al. Ibrutinib as Initial Therapy for 
Patients with Chronic Lymphocytic Leukemia. N.Engl.J.Med. 
2015;373:2425-2437. 
 
3. 
Byrd JC, Furman RR, Coutre SE et al. Three-year follow-up of treatment-
naive and previously treated patients with CLL and SLL receiving single-
agent ibrutinib. Blood 2015;125:2497-2506. 
 
4. 
Gribben JG. Stem-cell transplantation in chronic lymphocytic 
leukaemia. Best.Pract.Res.Clin.Haematol. 2007;20:513-527. 
 
5. 
Rai KR, Peterson BL, Appelbaum FR et al. Fludarabine compared with 
chlorambucil as primary therapy for chronic lymphocytic leukemia. 
N.Engl.J.Med. 2000;343:1750-1757. 
 
6. 
Johnson S, Smith AG, Loffler H et al. Multicentre prospective randomised trial of 
fludarabine versus cyclophosphamide, doxorubicin, and prednisone (CAP) for 
treatment of advanced-stage chronic lymphocytic leukaemia. The French 
Cooperative Group on CLL. Lancet 1996;347:1432-1438. 
 
7. 
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Appendix A. - CARTOX: CAR T-Cell Therapy Toxicity Assessment and Management 
 


 
 
 
 
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Appendix B. -  CARTOX: CTCAE Grading of Common Organ Toxicities 
 


 
 
 
 
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