Clinical trial NCT04097301
Conditions: Acute Myeloid Leukemia Multiple Myeloma
Sponsor: AGC Biologics S.p.A.
Start date: 2019-08-27; Completion date: 2021-06-18
Enrollment: 8 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this first-in-man Phase I-IIa study is to evaluate the safety and antitumor activity of autologous CD44v6 CAR T-cells in patients with acute myeloid leukemia (AML) and multiple myeloma (MM).

Detailed description:
The study is a seamless Phase I/IIa, open-label, multicenter clinical trial that combines Phase I dose escalation based on toxicity with Phase IIa dose expansion based on antitumor activity. Considering the "first in human" nature of this clinical study, the Bayesian Optimal Interval Design (BOIN) has been chosen to minimize any risks of exposure to the novel CD44v6 CAR T-cells during dose escalation. The study population is made up of patients with relapsed/refractory AML or MM expressing CD44v6.

The medicinal product under investigation (MLM-CAR44.1 T-cells) is patient specific as it is prepared starting from lymphocytes of the patient collected through lymphocyte apheresis. These autologous T-cells are expanded in vitro in large numbers and genetically modified to express the CAR CD44v6ΔNL gene and thus acquire antitumor functions. As a safety feature, the MLM-CAR44.1 T-cells are genetically modified to also express the HSV-TK Mut2 gene (suicide gene), which can be selectively activated in case of severe toxicity through the administration of ganciclovir (GCV), leading to the death of proliferating CAR T-cells.

The aim of this study is to assess the safety, antitumor activity and feasibility of CD44v6 CAR T cell immunotherapy in AML and MM.

Arms: arm 1: PHASE I: i.v. single dose of MLM-CAR44.1 T-cells: 0.5 x 10E6/Kg or 1 x 10E6/Kg or 2 x10E6/Kg according to the BOIN design.

PHASE IIa: i.v. single dose of MLM-CAR44.1 T-cells corresponding to the maximum tolerated dose (MTD).

Phase I and IIa Pre-treatment: lymphodepleting chemotherapy with cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) daily from day -5 to day -3
Interventions: intervention 1: MLM-CAR44.1 T-cells at day 0 Single intravenous infusion
Intervention descriptions: intervention 1: Lymphodepleting chemotherapy with cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) daily from day -5 to day -3.

Protocol documents:
Prot_SAP_000.pdf: 


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Synopsis of the Protocol 
 
Title 
A Phase I-IIa trial to assess the safety and antitumor  activity of autologous 
CD44v6 CAR T-cells in acute myeloid leukemia and multiple myeloma 
expressing CD44v6
Version  
C 
Sponsor 
MolMed S.p.A.
Indication 
Acute Myeloid Leukemia and Multiple Myeloma
Objectives 
Primary objectives of Phase I 
1. To determine the maximum tolerated dose (MTD) and recommended 
phase IIa dose of MLM-CAR44.1 T-cells in patients with 
relapsed/refractory acute myeloid leukemia (AML) or multiple 
myeloma (MM) expressing CD44v6. 
2. To evaluate the overall safety of treatment with MLM-CAR44.1 T-cells.
3. To monitor for the absence of replication-competent retrovirus (RCR) 
Secondary objectives of Phase I 
1. To evaluate hematological response to MLM-CAR44.1 T-cells in AML 
and MM. 
2. To characterize the in vivo pharmacokinetic profile (engraftment, 
persistence, trafficking) of MLM-CAR44.1 T-cells. 
3. To assess suicide gene activation and elimination of transduced cells 
through administration of ganciclovir in case of cytokine release 
syndrome (CRS) or other MLM-CAR44.1 T-cell related toxicities. 
4. To conduct an ancillary study, including but not limited to the 
characterization of the cellular composition of circulating LNGFR+ 
(CAR T-cells) and LNGFR- cells, and the monitoring of the patient’s 
immune status through analyses of peripheral blood mononuclear cells 
(PBMC). 
5. To set-up a validation process among the participating centers for the 
CD44v6 marking of tumor cells (AML and MM).  
Primary objective of phase IIa 
To evaluate hematological response to MLM-CAR44.1 T-cells in AML and 
MM. 
Secondary objectives of Phase IIa 
1. To evaluate the antitumor activity of MLM-CAR44.1 T-cells 
2. To characterize the in vivo pharmacokinetic profile (engraftment, 
persistence, trafficking) of MLM-CAR44.1 T-cells. 
3. To evaluate the feasibility of treatment with MLM-CAR44.1 T-cells  
4. To evaluate the safety of MLM-CAR44.1 T-cells. 
5. To conduct an ancillary study, including but not limited to the 
characterization of the cellular composition of circulating LNGFR+ 
(CAR T-cells) and LNGFR- cells, and the monitoring of the patient’s 
immune status through analyses of PBMCs. 


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Exploratory objectives of Phase IIa 
To evaluate minimal residual disease
Study design 
This is a seamless Phase I/IIa, open-label, multicenter clinical trial that 
combines Phase I dose escalation based on toxicity with Phase IIa dose 
expansion based on antitumor activity.  
Phase I dose escalation 
A Bayesian Optimal Interval (BOIN) design with cohorts of 3 patients for each 
indication (AML and MM) will be used to single out the MTD based on a target 
toxicity rate of 30% and an interval of 25% - 35%. 
The first AML cohort and first MM cohort will be made up of adult patients 
and will be treated with the lowest dose of autologous MLM-CAR44.1 T-cells 
(0.5 x 10E6/Kg). The first three enrolled patients (regardless of indication) will 
be treated one at a time at intervals of 36 days to evaluate toxicity. These three 
patients will undergo lymphodepleting chemotherapy prior to CAR T-cell 
infusion only once the previously enrolled patient has completed the 30-day 
follow-up after CART T-cell infusion. The remaining patients of the first 
cohorts will be treated without staggering once the first three enrolled patients 
have completed follow-up and the dose has proven to be safe. After all patients 
of the first AML cohort or first MM cohort have completed the 30-day safety 
monitoring period, the decision on whether to escalate to the next dose will be 
made together with a Data and Safety Monitoring Board (DSMB) based on the 
rate of dose-limiting toxicities (DLT) and the decision rules presented in the 
Table below. The second and then third dose will be administered to cohorts 
of new patients as outlined for dose 1 with an escalation process that continues 
until the MTD or the maximum sample size of 15 patients for each indication 
has been reached. The duration of the evaluation period for each patient (30 
days) is based on previous studies with similar cellular products, in which 
severe cytokine release syndrome (CRS) (main DLT for the study) was 
observed for up to 7-15 days after infusion. 
0.5 x 10E6/Kg, 1 x 10E6/Kg and 2 x 10E6/Kg are the three doses of autologous 
MLM-CAR44.1 T-cells for this study. The recommended Phase IIa dose 
corresponds to the MTD. If no DLT is reported, the dose will be the maximum 
dose investigated (2 x 10E6/kg). 
The BOIN design is a novel Bayesian dose-finding method that minimizes the 
decision errors of dose assignment (for example the chance of exposing 
patients to overly toxic doses) and thus safeguards patient ethics and safety by 
allowing decisions to be made at any time during the study, even when one of 
the patients in the cohort is not evaluable. This design has performance superior 
to the traditional 3 + 3 design but shares with it the fact that it can be easily 
implemented, differently from the well-known continual reassessment method 
(CRM), the gold standard of Phase I dose finding designs. The BOIN design 
yields an average performance comparable with CRM in selecting the MTD 
but has a lower risk of assigning patients to sub therapeutic or overly toxic 
doses. 
 
 
 
 
 


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Transition rules for each dose 
Number 
of 
patients 
with at 
least 
one 
DLT 
Cumulative Number of Patients 
  
3 
6 
9 
12 
15 
0 
E 
E 
E 
E 
E 
1 
S 
E 
E 
E 
E 
2 
D 
S 
E 
E 
E 
3 
DU 
D 
S 
S 
E 
4 
  
DU 
D 
S 
S 
5 
  
DU 
DU 
D 
S 
6 
  
DU 
DU 
D 
D 
7 
  
  
DU 
DU 
D 
8 
  
  
DU 
DU 
DU 
9 
  
  
DU 
DU 
DU 
10 
  
  
  
DU 
DU 
11 
  
  
  
DU 
DU 
12 
  
  
  
DU 
DU 
13 
  
  
  
  
DU 
14 
  
  
  
  
DU 
15 
  
  
  
  
DU 
E: Escalate to the next higher dose; S: Stay at the same dose; D: De‐escalate to the previous lower dose; 
DU: De‐escalate to the previous lower dose and the current dose will never be used again in the trial 
(safety rule).  
Phase IIa dose expansion 
The activity of the MTD will be assessed on 14 patients of each indication 
(AML and MM separately) using Simon’s two-stage design. These cohorts will 
include the patients enrolled in Phase I treated with the MTD, and in the case 
of AML, also children (except for Czech Republic (CZ) site). 
In the first stage, the hematologic response to treatment of the first 6 patients 
with each indication will be evaluated, and if no response is observed the study 
will be terminated. Otherwise, the study will proceed to stage two and the 
remaining 8 subjects will be enrolled.  
Patients with either indication will be followed up for 24 months to evaluate 
the antitumor activity, safety and feasibility of treatment with MLM-CAR44.1 
T-cells. 
Transition from Phase I to Phase II will be managed through the submission 
of a substantial amendment to the protocol, which will be evaluated in 
consultancy with the Data Safety Monitoring Board (DSMB). 
Number of patients 
The study will be conducted in 5 sites in Italy, Spain, Germany and Czech 
Republic. 
During Phase I, the study will enroll up to 15 adult patients with AML and up 
to 15 adult patients with MM. Phase IIa of the study will evaluate newly 
enrolled patients, including children with AML, and patients who were treated 
at the MTD during Phase I for a combined maximum of 14 patients per 
indication. 
The trial will include pediatric patients (except for CZ site) with AML (˂ 18 
years old) only in Phase IIa and if all the following criteria have been met by 
adult patients in Phase I: 
1. No SAEs related to lymphocyte apheresis. 
2. No immediate SAEs related to MLM-CAR44.1 T-cell infusion (within 48 
hours of infusion). 


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3. No early SAEs related to infusion (within 30 days). 
4. Evidence of MLM-CAR44.1 T-cell engraftment. 
Should one or more of these criteria not be met, the DSMB (including a 
pediatrician) will evaluate the risk/possible benefit profile of study treatment 
and give recommendations on whether to include pediatric patients.  
The protocol will be amended to classify eligible pediatric patients according 
to age groups. 
Study population 
The study population is made up of patients with relapsed/refractory AML or 
MM expressing CD44v6. 
Inclusion criteria 
1. Written informed consent before any study-related procedure. 
2. Adults and children 
a) Adults 18 to 75 years old with AML or MM or 18 to 63 years old for 
CZ site 
b) Children 1 to 17 years old with AML, only in Phase IIa, except for CZ 
site 
3. Confirmed diagnosis of AML or MM as follows: 
a) AML: Primary or secondary AML (any subtype except acute 
promyelocytic leukemia) according to World Health Organization 
(WHO) classification 
b) MM with measurable disease defined at least one of the following: 
 Serum M protein ≥5 g/dL (≥5 g/L);  
 Urine M protein ≥100 mg/24 hours;  
 
abnormal serum free light chain (sFLC) assay: FLC ratio <0.26 or 
>1.65 and/or involved Serum free light chain >=100 mg/L 
4. Patients with relapse or refractory disease: 
a) AML patients must be unlikely to benefit from cytotoxic 
chemotherapy as follows:  
 Leukemia refractory to at least 2 induction attempts (use of a 
hypomethylating agent for at least 4 cycles can be considered a 
line of treatment). 
 Leukemia in relapse within 1 year following complete response 
(CR) after at least 2 induction attempts (use of a hypomethylating 
agent for at least 4 cycles can be considered a first line of 
treatment). 
 High-risk leukemia in adults according to 2017 European 
LeukemiaNet (ELN) in first relapse after a hypomethylating agent 
or a cycle containing cytarabine at a dose ≥ 1g/sqm a day (e.g. 
FLAG-IDA), except for FLT3-mutated AML. 
 High-risk leukemia in children as defined by the Italian 
Association of Pediatric Hematology and Oncology (AIEOP). 
b) Patients with MM must have a relapse or refractory disease after at 
least 4 different prior treatments in 3 treatment lines, or 4 treatments 
in 2 treatment lines in case of early relapsing patients (relapse in less 
than 1.5 years). Treatments include: 
 Proteasome inhibitor  


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 High-dose alkylating agent if patient less than 70 years old 
 Immunomodulatory drug (IMID) 
 A monoclonal antibody (i.e. anti CD38 monoclonal antibody) 
5. Positive CD44v6 expression on tumor cells by flow cytometry. 
6. Eastern Cooperative Oncology Group (ECOG) performance status 0-2. 
7. Life expectancy of at least 12 weeks. 
8. Adequate organ function: 
a) Alanine aminotransferase (ALT) level within 2.5 times the 
institutional upper limit of normal (ULN). 
b) aspartate aminotransferase (AST) level ≤ 2.5 x ULN.  
c) Total bilirubin level ≤ 1.5 x ULN, or ≤ 2.5 x ULN in case of history of 
Gilbert’s disease. 
d) Serum creatinine ≤ 2.0 mg/dL or a calculated or measured creatinine 
clearance ≥ 45mL/min.  
e) Corrected Diffusing Capacity of Carbon Monoxide (DLCO) (via 
Dinakara Equation) or FEV1 of  66% without dyspnea on slight 
activity after hemoglobin correction. 
f) Left ventricular ejection fraction ˃ 45%. 
9. Recovery from toxicities of clinical consequence attributed to previous 
chemotherapy to CTCAE v5.0 Grade 1 (i.e., certain toxicities such as 
alopecia will not be considered in this category).  
10. Ability to comply with study procedures, including hospitalization and 
protocol-specified acquisition of blood and/or bone marrow specimens. 
11. Willing to be followed up long-term (15 years) as required by health 
authorities for cell and gene therapy products 
12. Women of childbearing potential must have test negative for pregnancy at 
enrolment and during the study and agree to use an effective method of 
contraception.  
Exclusion criteria 
1. History of or candidate for allogeneic stem cell transplantation. 
2. Cardiovascular, pulmonary, renal, and hepatic functions that in the 
judgment of the investigator are insufficient to undergo investigational 
CAR T-cell therapy. 
3. Any history of or suspected current autoimmune disorders (apart from 
vitiligo, resolved childhood atopic dermatitis, Graves’ disease clinically 
controlled).  
4. History of rheumatologic disorders requiring specific treatment at any time 
in the patient’s medical history. 
5. Second primary malignancy that requires active therapy. Adjuvant 
hormonal therapy is allowed.  
6. Known or suspected central nervous system (CNS) leukemia.  
7. Presence or history of myeloid sarcoma or any extramedullary mass. 
8. Any medical or psychiatric condition that may limit compliance or 
increase safety risks, such as:  
a) Active uncontrolled infection (including, but not limited to viral, 


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bacterial, fungal, or mycobacterial infection). 
b) Patients with known multiple antibiotic resistant infections in their 
clinical history. 
c) Known human immunodeficiency virus infection, active or chronic 
hepatitis B or C infection.  
d) Grade 3 or 4 bleeding.  
e) Uncontrolled hypertension (systolic pressure > 180 mm Hg or diastolic 
pressure > 100 mm Hg).  
f) Clinically significant arrhythmia, clinically significant baseline QTcF, 
or QTcF > 480 msec.  
g) Unstable angina.  
h) Myocardial infarction within 6 months prior to study entry.  
i) Clinically significant heart disease (e.g. CHF NYHA III or IV, 
unstable coronary artery disease, myocardial infarction < 6 mo. prior 
to study entry). 
j) Pregnancy or breast feeding.  
k) Major surgery or trauma within 4 weeks before enrollment.  
l) Dementia or altered mental status that would preclude sufficient 
understanding to provide informed consent. 
Once all the above eligibility criteria are confirmed, a lymphocyte apheresis 
product of non-mobilized cells must be received and accepted by the 
manufacturing site. Note: the lymphocyte apheresis product will not be shipped 
or assessed for acceptance by the manufacturing site until documented 
confirmation of all other inclusion/exclusion criteria have been satisfied. 
Prior to lymphocyte apheresis (week -9 to -7), the following criteria must be 
met: 
1. Peripheral blast count ≤ 20,000/mm3 (AML). 
2. No treatment with any other investigational agent in the previous 4 weeks. 
3. No treatment with an immunostimulatory agent (IMIDs are allowed) or 
any cell therapy in the previous 30 days. 
4. Negative to the following tests: HCV (Antibody, NAT), HIV 1-2 (p24, AB, 
Ag and NAT), total Ig Treponema Pallidum (if positive perform specific 
test), Australia HBsAg, total anti HB core Ab (if positive perform HBV 
DNA NAT), mycoplasma (PCR or IgM) and HTLV I-II. 
Prior to lymphodepleting chemotherapy and MLM-CAR44.1 T-cell 
infusion (day -5 to day -3), the following criteria must be met: 
1. Evidence of active disease at the beginning of lympho-depleting 
chemotherapy.  
2. The following medications are excluded and should not be administered 
concomitantly or following lymphodepleting chemotherapy:  
a) Monoclonal antibodies in the 8 weeks prior to MLM-CAR44.1 T-cell 
infusion are prohibited. 
b) Salvage chemotherapy (e.g. clofarabine, cytosine arabinoside > 100 
mg/m2, anthracyclines, cyclophosphamide, proteasome inhibitors, 
IMIDs) must be stopped > 2 weeks prior to lymphodepleting 
chemotherapy 


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c) Granulocyte colony stimulating or granulocyte-macrophage colony 
stimulating factor in the 2 weeks prior to study CAR T-cell 
administration. 
d) Immunosuppressant medications in the 2 weeks prior to CAR T-cell 
administration. 
e) Cytosine arabinoside < 100 mg/m2/day must be stopped > 1 week prior 
to MLM-CAR44.1 T-cell infusion. 
f) Therapeutic systemic doses of steroids must be stopped > 72 hours 
prior to product infusion. However, < 12 mg/m2/day hydrocortisone or 
equivalent are allowed as physiological replacement doses of steroids.
g) Hydroxyurea must be stopped > 72 hours prior to MLM-CAR44.1 T-
cell infusion. 
3. No cardiovascular, pulmonary, renal, and hepatic  functions that in the 
judgment of the investigator are insufficient to undergo investigational 
CAR T-cell therapy. 
4. Female patients of childbearing potential must have a negative pregnancy 
test within 24 hours prior to starting lymphodepleting therapy 
Study procedures 
Screening and Enrolment (Week -10 to Week -8) 
After providing written informed consent, patients with AML or MM will be 
screened, and those compliant with inclusion/exclusion criteria including 
CD44v6 expression on tumor cells will be enrolled in the study. Children with 
AML will be enrolled in Phase IIa only, except for CZ site 
In Phase I, the first three enrolled patients (regardless of indication) for each 
dose will be treated one at a time at intervals of at last 36 days. These three 
patients will be enrolled accordingly once the previously enrolled patient has 
been approved for lymphodepleting chemotherapy. The remaining patients for 
each dose will be enrolled once the dose has proven to be safe in the first three 
enrolled patients. 
Pretreatment and treatment  
 Lymphocyte apheresis (Week -9 to Week-7) 
The first step in manufacturing CAR T-cells is to collect autologous 
lymphocytes by apheresis. Patients will undergo non-stimulated lymphocyte 
apheresis at least 7 weeks prior to MLM-CAR44.1 T-cell infusion to allow cell 
production, quality and release controls, and completion of lymphodepleting 
chemotherapy. For an adequate cell collection, apheresis will take place before 
lymphodepleting chemotherapy. Patients may be required to undergo multiple 
apheresis procedures so a sufficient number of CD3+ cells (approximately 7 x 
10E8) or, if not possible, a sufficient number of WBC (approximately 10 x 
10E9) can be collected for the preparation of the investigational product. 
 Lymphodepleting chemotherapy (Day -5 to Day -3) 
Lymphodepleting chemotherapy prior to T-cell infusion has been shown to 
favor T-cell engraftment and proliferation. This preconditioning creates space 
for the expansion of infused cells, limits the competition for homeostatic 
gamma chain cytokines IL-7 and IL-15, depletes regulatory T cells, and 
activates the innate immune system. Patients will undergo lymphodepleting 
chemotherapy with cyclophosphamide (500 mg/m2) and fludarabine (30 
mg/m2) daily from day -5 to day -3. 


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 Treatment with MLM-CAR44.1 T-cell (Day 0) 
Patients will receive one dose of MLM-CAR44.1 T-cells during a single 
infusion. 
Follow-up 
After being infused with MLM-CAR44.1 T-cells, patients will be monitored 
closely in the hematology or pediatric ward for at least 15 days and then 
followed up twice weekly through Month 1, weekly for the second month, 
every two weeks for the third month, monthly until Month 6 and then every 3 
months until Month 24. 
Long-term follow-up 
A long-term follow-up is planned for assessing any potential delayed toxicity 
and lasting anti-tumor effects of MLM-CAR44.1 T-cells. Patients will 
continue to be followed for 15 years after MLM-CAR44.1 T-cell infusion as 
per health authority guidelines.
Product 
Frozen autologous T lymphocytes genetically modified ex vivo with a γ 
retroviral vector to express a mutated form of the Herpes Simplex Virus 
Thymidine Kinase (HSV‐TKmut2) and the CAR CD44v6ΔNL genes; 
formulated in freezing medium (7% HSA/10% DMSO in saline solution) at 
the final concentration of 1-10x10E6 cells/ml. 
0.5 x 10E6/Kg, 1 x 10E6/Kg and 2 x 10E6/Kg are the three doses of 
autologous MLM-CAR44.1 T-cells for this study
Comparator 
Not Applicable 
Endpoints 
Phase I 
Primary endpoints - safety 
1. MTD established through BOIN design and the following DLTs 
occurring within 30 days following CAR T-cell infusion: 
a. Grade 4 CRS 
b. Grade 3 or higher CRS not responsive to therapy with steroids and 
or tocilizumab/siltuximab within 24 hours 
c. Grade 3 or higher toxicity at least possibly related to treatment 
with MLM-CAR44.1 T-cells excluding hematological toxicities 
d. Skin toxicities:  
o NCI-CTC grade 3 or higher erythroderma (generalized 
exfoliative dermatitis) 
o Stevens-Johnson Syndrome 
o Toxic Epidermal Necrolysis (TEN) 
o Any other grade 3 or higher skin toxicity histologically 
confirmed as being related to CAR T-cell treatment 
e. Grade 3 or higher neurotoxicity 
2. Type, frequency and severity of adverse events and monitoring of 
systemic reactions (fever, tachycardia, nausea and vomiting, joint pain, 
skin rash) for 30 days following CAR T-cell infusion. 
3. Absence of replication competent retrovirus (RCR) monitored by 
DNA PCR for the Galv gene 3,6, 12 and 24 months after infusion and 
then yearly as defined in the long-term follow-up  
Secondary endpoints 
1. Hematologic disease response 


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a. AML: complete response (CR), incomplete response (CRi) 
and partial response (PR) according to ELN criteria 1 and 2 
months following MLM-CAR44.1 T-cell infusion.  
b. MM: overall response rate (ORR): stringent complete 
response (sCR), CR, very good partial response (VGPR) and 
partial response (PR) according to IMWG criteria 1 and 3 
months following T-cell infusion 
2. Levels of circulating MLM-CAR44.1 T-cells by flow cytometry. 
3. Suicide gene activation and elimination of transduced cells through 
administration of ganciclovir in case of cytokine release syndrome or 
other significant MLM-CAR44.1 T-cell related toxicities. 
4. To set-up a validation process among the participating centers for the 
CD44v6 marking of tumor cells (AML and MM).  
 
Phase IIa 
Efficacy 
Primary endpoint – hematologic disease response 
• 
AML: CR, CRi and PR rate as per ELN criteria, 2 months after MLM-
CAR44.1 T-cell infusion. 
• 
MM: ORR: (sCR, CR, VGPR, and PR) as per IMWG criteria, 3 
months after T-cell infusion. 
Secondary endpoints 
1. Hematologic disease response: 
a. AML: CR, CRi and PR rate as per ELN criteria, 1 month and 
6 months after MLM-CAR44.1 T-cell infusion. Morphologic 
leukemia-free state (MLFS) 1, 2 and 6 months after MLM-
CAR44.1 T-cell infusion. 
b. MM: ORR (sCR, VGPR and PR) as per IMWG criteria, 1,2 
and 6 months after T-cell infusion. 
2. Overall Survival (OS) at 2 years: time from MLM-CAR44.1 T-cell 
infusion to death due to any cause. 
3. Disease-Free Survival (DFR) in AML patients: time from achievement 
of response to relapse or death due to any cause during response. 
4. Event Free Survival (EFS) in AML patients: time from the date of 
MLM-CAR44.1 T-cell infusion to the date of the earliest of the 
following: last follow-up, resistance, relapse or death due to any cause. 
5. Progression Free Survival (PFS) in MM patients at 2 years: time from 
MLM-CAR44.1 T-cell infusion to progression/relapse or death due to 
any cause.  
6. Duration of Remission (DOR) in MM patients: time from achievement 
of response to relapse or death due to any cause. 
Exploratory endpoint 
Minimal Residual Disease: 
• 
AML: proportion of patients with a molecular CR, 2 months after 
infusion. 
• 
MM: proportion of patients with a molecular CR, 3 months after 
infusion. 
Pharmacokinetics 
Levels of circulating MLM-CAR44.1 T-cells by flow cytometry. 


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Feasibility 
1. Percentage of eligible patients for each indication after initial 
screening who undergo both lymphocyte apheresis and CAR T-cell 
infusion. 
2. Percentage of patients who are eligible for lymphocyte apheresis and 
are then effectively infused with CAR T-cells. 
Safety 
1. Adverse events (any grade) and monitoring of systemic reactions 
(fever, tachycardia, nausea and vomiting, joint pain, skin rash). 
2. Absence of RCR monitored by DNA PCR for the Galv gene 3, 6, 12 
and 24 months after infusion and then yearly as defined in the long-
term follow-up. 
3. Absence of abnormal MLM-CAR44.1 T-cell clonal expansion 
monitored by clinical and laboratory surveillance, as well as by TCR-
Vbeta repertoire study 6 months after infusion. 
4. Suicide gene activation and elimination of transduced cells through 
administration of ganciclovir in case of cytokine release syndrome or 
other significant MLM-CAR44.1 T-cell related toxicities. 
Pharmacokinetics/Pha
rmacodynamics  
Pharmacokinetics: Levels of circulating MLM-CAR44.1 T-cells by flow 
cytometry 
Quality of life 
Not Applicable 
Statistical analysis 
Phase I 
Primary endpoints – safety 
a. The proportion of patients with DLTs occurring within one month 
from CAR T infusion will be calculated for each dose level along with 
the 95% exact Clopper-Pearson confidence intervals. 
b. The incidence of adverse events and of systemic reactions for 30 days 
following CAR T-cell infusion will be tabulated by MedDRA 
preferred term, system organ class and severity. 
c. The proportion of patients with absence of replication competent 
retrovirus monitored by DNA PCR at 3, 6, 12 and 24 months after 
infusion of MLM-CAR44.1 T-cells will be calculated for each dose 
level. The corresponding 95% exact Clopper-Pearson confidence 
intervals will also be calculated. 
Secondary endpoints 
a. Hematologic disease response for AML 
The percentages of patients either in CR, Cri or PR (ELN criteria) at 1 
and 2 months following MLM-CAR44.1 T-cell infusion will be 
estimated at each dose level and the 95% exact Clopper-Pearson 
confidence intervals calculated. 
b. Hematologic disease response for MM 
The overall response rate (ORR) is defined as the proportion of patients 
in: stringent complete response (sCR), CR, very good partial response 
(VGPR) and partial response (PR) (IMWG criteria). The percentages 
of overall response at 1 and 3 months following MLM-CAR44.1 T-
cell infusion will be estimated at each dose level and the 95% exact 
Clopper-Pearson confidence intervals calculated. 
c. Suicide gene activation  
The percentage of patients for whom activation of suicide gene was 


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needed will be calculated for each dose level along with the 
corresponding 95% exact Clopper-Pearson confidence intervals. 
The levels of transduced cells in patients treated with ganciclovir will 
be listed and described at each dose level.  
d. In vivo pharmacokinetic profile  
The pharmacokinetic analysis will be provided in the statistical 
analysis plan. 
Phase IIa 
Primary endpoints 
a. Hematologic disease response for AML at 2 months 
The percentages of patients in CR, Cri and PR (ELN criteria) at 2 
months following MLM-CAR44.1 T-cell infusion will be estimated, 
and the 95% exact Clopper-Pearson confidence intervals calculated. 
b. Hematologic disease response for MM at 3 months 
ORR: the percentages of overall response at 3 months following 
MLM-CAR44.1 T-cell infusion will be estimated, and the 95% exact 
Clopper-Pearson confidence intervals calculated. 
Secondary/exploratory endpoints: 
Efficacy 
a. CR, Cri, PR and morphologic leukemia-free state rates as per ELN 
criteria, 1 month and 6 months after MLM-CAR44.1 T-cell infusion 
(AML) will be summarized and presented with 95% confidence 
intervals. 
b. ORR (sCR, VGPR and PR) as per IMWG criteria, 1, 2 and 6 months 
and 6 months after T-cell infusion (MM) will be summarized and 
presented with 95% confidence intervals. 
c. OS, DFS (AML patients), EFS (AML patients) DoR (MM patients), 
and PFS (MM patients) will be estimated using the Kaplan-Meier 
product-limit method. Median and two-sided 95% confidence intervals 
for median values will be computed and Kaplan-Meier plots will be 
presented. 
d. Minimal Residual Disease for AML: The percentages of patients with 
a molecular CR at 2 months for AML and at 3 months for MM will be 
estimated and the 95% exact Clopper-Pearson confidence intervals 
calculated. 
Feasibility 
a. The percentages of eligible patients who undergo both lymphocyte 
apheresis and CAR T-cell infusion will be estimated, and the 95% 
exact Clopper-Pearson confidence intervals calculated. 
b. The percentages of eligible patients effectively infused with CAR T-
cells after undergoing lymphocyte apheresis will be estimated, and the 
95% exact Clopper-Pearson confidence intervals calculated.  
Safety 
a. SAEs, treatment-emergent AEs and systemic reactions will be 
summarized for each dose level by system organ class and/or preferred 
term and severity by means of frequency tables.  


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b. The proportion of patients with absence of replication competent 
retrovirus monitored by DNA PCR at 3, 6 and 12 and 24 months after 
infusion of MLM-CAR44.1 T-cells will be calculated. The 
corresponding 95% exact Clopper-Pearson confidence intervals 
calculated. 
c. The proportion of patients with absence of abnormal MLM-CAR44.1 
T-cell clonal expansion monitored by clinical and laboratory 
surveillance, as well as by TCR-Vbeta repertoire study will be 
calculated 6 months after infusion. The corresponding 95% exact 
Clopper-Pearson confidence intervals will also be calculated. 
d. The percentage of patients for whom activation of suicide gene was 
needed will be calculated along with the corresponding 95% exact 
Clopper-Pearson confidence intervals. The levels of transduced cells 
in patients treated with ganciclovir will be listed and described.  
Pharmacokinetic profile 
The pharmacokinetic analysis will be provided in the statistical analysis plan. 
Ancillary study 
The ancillary study will be provided in the statistical analysis plan 
Validation process for the CD44v6 marking of tumor cells (AML and MM) 
The analysis will be provided in the statistical analysis plan 
Study duration 
48 months (the end of study is defined as the last patient’s last visit (LPLV), 
which is the last patient’s Month 24 evaluation, or the time of premature 
withdrawal 
 
 
 


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Table of contents 
 
1 
INTRODUCTION ........................................................................................................ 23 
 
Background .............................................................................................................. 23 
1.1.1 
Acute Myeloid Leukemia ...................................................................................................... 23 
1.1.2 
Multiple Myeloma ................................................................................................................. 23 
1.1.3 
Chimeric Antigen Receptor (CAR) T-Cells ......................................................................... 24 
1.1.4 
CD44v6 .................................................................................................................................. 26 
1.1.5 
EURE-CART Project ............................................................................................................ 27 
 
Drug description ...................................................................................................... 27 
 
Drug activity ............................................................................................................. 28 
1.3.1 
Non-clinical studies .............................................................................................................. 29 
1.3.2 
Clinical studies ...................................................................................................................... 31 
 
Rationale – risks and benefits ................................................................................. 31 
1.4.1 
Possible benefits of CAR-T cell therapy .............................................................................. 31 
1.4.2 
Possible risks of CAR T-cell Therapy .................................................................................. 32 
1.4.2.1 
Cytokine release syndrome.................................................................................................... 33 
1.4.2.2 
On-target/off tumor recognition ............................................................................................ 34 
1.4.2.3 
Off-target antigen recognition ............................................................................................... 35 
1.4.2.4 
Neurotoxicity ......................................................................................................................... 35 
1.4.2.5 
Allergic reactions and Tumor Lysis Syndrome (TLS) .......................................................... 36 
1.4.2.6 
Infectious diseases ................................................................................................................. 36 
1.4.2.7 
Insertional oncogenesis ......................................................................................................... 37 
1.4.3 
Suicide gene .......................................................................................................................... 37 
2 
OBJECTIVES ............................................................................................................... 38 
 
Phase I objectives ..................................................................................................... 38 
2.1.1 
Primary objectives ................................................................................................................ 38 
2.1.2 
Secondary objectives ............................................................................................................. 38 
 
Phase IIa objectives ................................................................................................. 39 
2.2.1 
Primary objective .................................................................................................................. 39 
2.2.2 
Secondary objectives ............................................................................................................. 39 
2.2.3 
Exploratory objectives .......................................................................................................... 40 
3 
TRIAL DESIGN ........................................................................................................... 41 
 
Phase I dose escalation ............................................................................................ 41 
 
Phase IIa dose expansion ........................................................................................ 43 
 
Follow-up .................................................................................................................. 43 
 
End of study ............................................................................................................. 43 


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4 
PATIENT SELECTION CRITERIA ......................................................................... 46 
 
Target population .................................................................................................... 46 
 
Inclusion criteria ...................................................................................................... 46 
 
Exclusion criteria ..................................................................................................... 48 
4.3.1 
At screening .......................................................................................................................... 48 
 
Prior to lymphocyte apheresis (week -9 to -7). ...................................................... 49 
 
Prior to lymphodepleting chemotherapy/ MLM-CAR44.1 T-cell infusion (day -5 
to day -3) ................................................................................................................... 49 
5 
EXPERIMENTAL PRODUCT ................................................................................... 51 
 
Study drug information ........................................................................................... 51 
 
Study drug supply .................................................................................................... 51 
 
Study drug accountability, disposal and destruction ........................................... 51 
 
Formulation, packaging, labelling and storage ..................................................... 51 
 
Dosage schedule ....................................................................................................... 52 
5.5.1 
Dose modification ................................................................................................................. 52 
5.5.2 
Treatment duration ............................................................................................................... 52 
 
Lymphocyte apheresis ............................................................................................. 52 
 
Salvage chemotherapy ............................................................................................. 53 
 
Lymphodepleting chemotherapy ............................................................................ 53 
 
Treatment with MLM-CAR44.1 T-cells ................................................................ 54 
5.9.1 
Premedication ....................................................................................................................... 55 
5.9.2 
MLM-CAR44.1 T-cell infusion ............................................................................................ 55 
 
Rescue medication ................................................................................................... 56 
 
Concomitant therapy ............................................................................................... 57 
 
Stopping rules .......................................................................................................... 57 
5.12.1 
Criteria for interrupting or discontinuing the study ........................................................... 57 
5.12.1.1  Phase I ................................................................................................................................... 57 
5.12.1.2  Phase IIa ................................................................................................................................ 57 
6 
STUDY ASSESSMENTS AND PROCEDURES ....................................................... 59 
 
Acute Myeloid Leukemia ........................................................................................ 59 
6.1.1.1 
Re-screening .......................................................................................................................... 60 
6.1.1.2 
Evaluation of CD44v6 expression on tumor cells ................................................................. 60 
6.1.2 
Lymphocyte apheresis (Week -9 to Week -7) ....................................................................... 60 
6.1.3 
Pre-chemotherapy evaluation visit (Week -2) ...................................................................... 61 
6.1.4 
Lymphodepleting chemotherapy (Day -5 to Day -3) ........................................................... 61 
6.1.5 
Pre-infusion visit (Day -1) .................................................................................................... 61 
6.1.6 
MLM-CAR44.1 T-cell infusion (Day 0) ............................................................................... 62 
6.1.7 
Post-infusion visits: D4±1d, D7±1d, D11±1d, D14±1d, D18±1d, D21±1d, D±25±1d and 
D28±1d (Month 1) ................................................................................................................ 62 


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6.1.8 
Post-infusion visits: Weekly from W5±3d to W8±3d (Month 2) ......................................... 63 
6.1.9 
Post-infusion visit: W10±3d and W12±3d (Month 3) ......................................................... 63 
6.1.10 
Post-infusion visits: Monthly from M4±14d to M6±14d ..................................................... 64 
6.1.11 
Post-infusion visits: M9±14d, M12±14d, M15±14d, M18±14d, M21±14d, M24 ±14d (end of 
primary follow-up) ................................................................................................................ 64 
 
Multiple Myeloma .................................................................................................... 65 
6.2.1 
Screening (Week -10 to Week -8) and enrollment ............................................................... 65 
6.2.1.1 
Re-screening .......................................................................................................................... 66 
6.2.1.2 
Evaluation of CD44v6 expression on tumor cells ................................................................. 66 
6.2.2 
Lymphocyte apheresis (Week -9 to Week -7) ....................................................................... 67 
6.2.3 
Pre-chemotherapy evaluation visit (Week -2) ...................................................................... 67 
6.2.4 
Lymphodepleting chemotherapy (Day -5 to Day -3) ........................................................... 67 
6.2.5 
Pre-infusion visit (Day -1) .................................................................................................... 68 
6.2.6 
MLM-CAR44.1 T-cell infusion (Day 0) ............................................................................... 68 
6.2.7 
Post-infusion visits: D4±1d, D7±1d, D11±1d, D14±1d, D18±1d, D21±1d, D±25±1d, D28±1d 
(Month 1) .............................................................................................................................. 69 
6.2.8 
Post-infusion visits: Weekly from W5±3d to W8±3d (Month 2) ......................................... 70 
6.2.9 
Post-infusion visit: W10±3d and W12±3d (Month 3) ......................................................... 70 
6.2.10 
Post-infusion visits: Monthly from M4±14d to M6±14d ..................................................... 71 
6.2.11 
Post-infusion visits: M9±14d, M12±14d, M15±14d, M18±14d, M21±14d, M24 ±14d (end of 
primary follow-up) ................................................................................................................ 72 
 
End of primary follow-up and premature withdrawal ........................................ 72 
 
Secondary follow-up ................................................................................................ 73 
 
Long-term follow-up ................................................................................................ 73 
 
Quality of Life assessment ...................................................................................... 73 
 
Contraception ........................................................................................................... 74 
7 
SCHEDULE OF ASSESSMENTS .............................................................................. 75 
8 
EVALUATION CRITERIA ........................................................................................ 86 
 
Expression of CD44v6 on tumor cells – AML and MM patients’ study inclusion
 ................................................................................................................................... 86 
 
Maximum tolerated dose (Phase I) - AML and MM ............................................ 86 
 
Disease response (Phase I and Phase IIa) – AML ................................................. 86 
8.3.1 
Morphology ........................................................................................................................... 87 
8.3.2 
Tumor cell assessment by flow cytometry ............................................................................ 87 
8.3.3 
Minimal residual disease (MRD) in bone marrow .............................................................. 87 
8.3.4 
Extramedullary disease ........................................................................................................ 88 
8.3.5 
Molecular cytogenetics by means of fluorescence in situ hybridization (FISH) ............... 88 
 
Disease response (Phase I and Phase IIa) MM ..................................................... 88 
8.4.1 
M-protein Assessment by PEP and IFE .............................................................................. 89 
8.4.2 
Free Light Chain (FLC) Protein Assessment ...................................................................... 90 


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8.4.3 
Plasma cell count in bone marrow ....................................................................................... 90 
8.4.4 
Assessment of soft tissue plasmacytoma (STP) ................................................................... 90 
8.4.5 
Skeletal survey ...................................................................................................................... 91 
8.4.6 
Minimal residual disease (MRD) ......................................................................................... 91 
8.4.7 
Corrected calcium ................................................................................................................. 91 
8.4.8 
Cytogenetics by means of fluorescence in situ hybridization (FISH) ................................ 91 
8.4.9 
CD44v6 evaluation by flow cytometry .................................................................................. 91 
 
Safety assessments (AML and MM) ...................................................................... 91 
8.5.1 
Adverse Events and safety monitoring ................................................................................. 92 
8.5.2 
Physical examination ........................................................................................................... 92 
8.5.3 
Eastern Cooperative Oncology Group (ECOG) Performance Status ................................. 92 
8.5.4 
Vital signs.............................................................................................................................. 92 
8.5.5 
Height and weight ................................................................................................................. 92 
8.5.6 
Cardiac assessments ............................................................................................................. 92 
8.5.7 
DLCO or FEV1 ..................................................................................................................... 92 
8.5.8 
Pregnancy test ....................................................................................................................... 92 
8.5.9 
Replication-competent retrovirus (RCR) testing ................................................................. 92 
 
Laboratory tests ....................................................................................................... 93 
 
Pharmacokinetics (AML and MM) ........................................................................ 94 
 
Ancillary studies ...................................................................................................... 94 
9 
STATISTICAL CONSIDERATIONS ........................................................................ 96 
 
Statistical design ...................................................................................................... 96 
9.1.1 
Primary study variable ......................................................................................................... 96 
9.1.2 
Secondary/exploratory study variables ................................................................................ 97 
9.1.3 
Analysis populations ........................................................................................................... 101 
9.1.4 
Data Quality Assurance ..................................................................................................... 101 
 
Sample size ............................................................................................................. 102 
10 
DATA AND SAFETY MONITORING BOARD (DSMB) ..................................... 103 
 
DSMB roles and responsibilities .......................................................................... 103 
 
Composition of the DSMB .................................................................................... 103 
 
Organizational diagram and data flow ................................................................ 104 
 
DSMB governance ................................................................................................. 105 
11 
SAFETY 106 
 
Definitions ............................................................................................................... 106 
 
Criteria of evaluation ............................................................................................ 106 
 
Adverse events of special interest (AESI) ............................................................ 107 
 
Reporting procedures ............................................................................................ 107 
11.4.1 
Adverse Events .................................................................................................................... 107 
11.4.2 
Serious Adverse Events ...................................................................................................... 108 


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11.4.3 
DLTs .................................................................................................................................... 109 
 
Procedures to be followed in the event of pregnancy ......................................... 109 
 
Risk management plan .......................................................................................... 109 
11.6.1 
Safety monitoring ............................................................................................................... 109 
11.6.1.1  Patient monitoring after CAR-T-cell infusion ..................................................................... 109 
11.6.1.2  Cytokine release syndrome (CRS) ...................................................................................... 111 
11.6.1.3  CAR T-cell related encephalopathy syndrome (CRES) ...................................................... 114 
11.6.1.4  On-target/off tumor recognition .......................................................................................... 117 
11.6.1.5  Tumor lysis syndrome (TLS) .............................................................................................. 120 
11.6.1.6  Infections ............................................................................................................................. 120 
11.6.1.7  Uncontrolled T-cell proliferation ........................................................................................ 121 
11.6.1.8  Replication-competent retrovirus (RCR) testing. ................................................................ 121 
11.6.2 
Urgent Safety Measures (USM) ......................................................................................... 121 
12 
ETHICS AND GENERAL STUDY ADMINISTRATION ..................................... 123 
 
Ethical aspects ........................................................................................................ 123 
 
Independent Ethics Committees/Institutional Review Board ........................... 123 
 
Informed consent ................................................................................................... 123 
 
Conditions for modifying the protocol ................................................................. 124 
 
Conditions for terminating the study ................................................................... 124 
 
Study documentation: CRF and record keeping ................................................ 124 
 
Source documents and background data ............................................................ 125 
 
Audits and inspections ........................................................................................... 125 
 
Case Report Forms ................................................................................................ 125 
 
Monitoring the study ............................................................................................. 125 
 
Confidentiality of trial documents and subject records ..................................... 126 
 
Publication of data and protection of trade secrets ............................................ 126 
13 
REFERENCES............................................................................................................ 127 
 
 
 


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List of Tables 
 
TABLE 3-1 
TRANSITION RULES FOR EACH DOSE ........................................................................ 43 
TABLE 7-1 
SCHEDULE OF ASSESSMENTS - ACUTE MYELOID LEUKEMIA ................................. 75 
TABLE 7-2 
SCHEDULE OF ASSESSMENTS - MULTIPLE MYELOMA ............................................ 80 
TABLE 7-3 
SCHEDULE OF ASSESSMENTS – SECONDARY FOLLOW-UP (AML AND MM) ......... 85 
TABLE 8-1 
DISEASE ASSESSMENTS AML .................................................................................... 87 
TABLE 8-2 
DISEASE ASSESSMENTS MM ...................................................................................... 88 
TABLE 8-3 
LOCAL CLINICAL LABORATORY ASSESSMENTS ....................................................... 93 
TABLE 8-4 
CENTRAL LABORATORY ASSESSMENTS .................................................................... 93 
TABLE 11-1 
SUPPORTIVE-CARE CONSIDERATIONS FOR CAR-T-CELL THERAPY .................... 110 
TABLE 11-2 
GRADING OF CYTOKINE-RELEASE SYNDROME (CRS) ........................................... 112 
TABLE 11-3 
CRS MANAGEMENT ALGORITHM .......................................................................... 113 
TABLE 11-4 
RECOMMENDATIONS FOR MANAGEMENT OF CAR-T-CELL CRES ...................... 116 
TABLE 11-5 
SEVERITY-OF-ILLNESS SCORE FOR TOXIC EPIDERMAL NECROLYSIS (SCORTEN)
 
119 
 
 
 


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List of Appendices 
Appendix 1: BOIN design ................................................................................................................. 132 
Appendix 2: International Staging System (ISS) and revised-ISS (R-ISS) for multiple myeloma
 ........................................................................................................................................................ 135 
Palumbo et al. 2015 ........................................................................................................................... 135 
Appendix 3: European LeukemiaNet (ELN) Response Criteria for Acute Myeloid Leukemia. 136 
Appendix 4: Modified International Myeloma Working Group (IMWG) Uniform Response 
Criteria for Multiple Myeloma ................................................................................................... 138 
Appendix 5: WHO classification of myeloid leukemia .................................................................. 140 
Appendix 6: ECOG performance status scale ................................................................................ 141 
Appendix 7: Grading of CAR-T-cell-related encephalopathy syndrome (CRES) ...................... 142 
Appendix 8: Common terminology criteria for Adverse Events .................................................. 143 
Appendix 9: 2017 European LeukemiaNet (ELN) recommendations for diagnosis and 
management of AML ................................................................................................................... 144 
Appendix 10: Grant members .......................................................................................................... 148 
Appendix 11: Amendment history ................................................................................................... 149 
 
 
 


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List of Abbreviations 
 
ACT 
Adoptive Cell Therapy 
AIEOP 
Italian Association of Pediatric Hematology and Oncology 
ALC 
Absolute Lymphocyte Count 
ALT 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Count 
AST 
Aspartate Aminotransferase 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
ALL 
Acute Lymphoblastic Leukemia 
AML 
Acute Myeloid Leukemia 
ASCT 
Allogeneic Stem Cell Transplantation 
BBB 
Blood-Brain Barrier 
BCMA 
B-Cell Maturation Antigen 
BOIN 
Bayesian Optimal Interval 
CAR 
Chimeric Antigen Receptor 
CEA 
Carcinoembryonic Antigen 
CMML 
Chronic Myelomonocytic Leukemia 
CR 
Complete Response 
CRA 
Clinical Research Associate 
Cri 
Incomplete response 
CRS 
Cytokine Release Syndrome 
CZ 
Czech Republic 
DFS 
Disease-Free Survival 
DLCO 
Corrected Diffusing Capacity of Carbon Monoxide 
DLT 
Dose Limiting Toxicity 
DOR 
Duration of Response 
DSMB 
Data and Safety Monitoring Board 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report Form 
FEV1 
Forced Expiratory Volume in 1 second 
FLC 
Free Light Chain 
Galv 
Gibbon ape leukemia virus 
GCV 
Ganciclovir 
GvHD 
Graft versus Host Disease 
GvT 
Graft versus Tumor 
HRQoL 
Health-Related Quality of Life 
HSC 
Hematopoietic Stem Cells 
HSV-TK 
Herpes Simplex Virus-Thymidine Kinase 
ICF 
Informed Consent Form 
IEC 
Independent Ethics Committee 
IFE 
Immunofixation Electrophoresis 
IMID 
Immune Modulating Drug 
IRB 
Institutional Review Board 
JMML 
Juvenile Myelomonocytic Leukemia 
LFS 
Leukemia Free Survival 
LNGFR 
Low Affinity Nerve Growth Factor Receptor 
LPLV 
Las Patient Last Visit 
LSC 
Leukemia Stem Cells 
Mab 
Monoclonal Antibodies 
MLFS 
Morphologic Leukemia-Free State 
MM 
Multiple Myeloma 
MNC 
Mononuclear Cell 
MRD 
Minimal Residual Disease 


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MTD 
Maximum Tolerated Dose 
NCI-CTC 
National Cancer Institute Common Terminology Criteria 
ORR 
Overall Response Rate 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells  
PCR 
Polymerase Chain Reaction 
PD 
Progressive Disease 
PEP 
Protein Electrophoresis 
PFS 
Progression Free Survival 
PR 
Partial Response 
RCR 
Replication Competent Retrovirus 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
ScFV 
Single-chain Variable Fragment 
sCR 
Stringent Complete Response 
SD 
Stable Disease 
SFLC 
Serum Free light chain protein assessment 
SIFE 
Serum Immunofixation 
SJS 
Stevens-Johnson Syndrome 
SPEP 
Serum Protein Electrophoresis 
STP 
Soft Tissue Plasmacytoma 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TAA 
Tumor Associated Antigen 
TLS 
TEN 
Tumor Lysis Syndrome 
Toxic Epidermal Necrolysis (TEN) 
UAR 
Unexpected Adverse Reaction 
ULN 
Upper Limit of Normal 
UIFE 
Urine Immunofixation 
UPEP 
Urine Protein Electrophoresis 
USM 
Urgent Safety Measures 
VCV 
Valganciclovir 
VGPR 
Very Good Partial Response 
WHO 
World Health Organization 
 
 
 


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1 
INTRODUCTION 
 Background 
1.1.1 Acute Myeloid Leukemia 
Relapsed or refractory acute myeloid leukemia (AML) poses a serious challenge to current 
therapeutic strategies. Of patients who are fit enough to receive standard induction therapy, 
about 60-80% of younger adults and 40-50% of older adults achieve a complete remission, 
leaving a substantial population of patients who are refractory to initial induction therapy 
(Mangan et al., 2011). An additional large population of patients have disease relapses after 
achieving first complete response (CR). In a study of 1,069 patients achieving a first CR at MD 
Anderson Cancer Center between 1991 and 2003 without undergoing Hematopoietic Stem Cell 
Transplantation (HSCT), the probability of relapse-free survival at 3 years was only 29% 
(Yanada et al., 2007). This has been attributed to leukemia stem cells (LSCs), which hide in 
endosteal and sinusoidal niches in the bone marrow and evade immune surveillance through 
the outgrowth of poorly immunogenic tumor cell variants, known as immune selection, and/or 
through disruption of the immune system. A robust innate immune system is therefore 
mandatory to avoid relapses by targeting chemoresistant malignant cells (Nwajei F et al., 2013).  
Especially in fit patients that suffer of intermediate or high-risk AML and achieve CR after 
induction therapy, HSCT remains the most effective long-term treatment, yielding cure in 50–
60% of patients (Cormelissen et al., 2015; Passweg et al., 2016). Nevertheless, several patients 
never become eligible for transplant because of co-morbidities, failure to reach a CR or lack of 
a suitable donor. Furthermore, available data suggest that HSCT may not be the most effective 
strategy to eradicate minimal residual disease (MRD) and that novel agents such as molecularly 
targeted drugs (FLT3 or IDH inhibitors) or monoclonal antibody–based agents including 
antibody-drug conjugates and bispecific antibodies, and, potentially, checkpoint inhibitors and 
CAR T-cells, may improve therapeutic strategies to eradicate persistent minimal residual 
disease remaining after cytotoxic regimens or can be used to “bridge” to transplant (Medlinger 
et al., 2016). 
1.1.2 Multiple Myeloma 
Multiple myeloma is the second most commonly diagnosed hematological neoplasm, with an 
incidence rate of 6.2 per 100,000 individuals (Seigel et al., 2016). In 2016, there were an 
estimated 30,330 newly diagnosed cases in the United States, accounting for ~1.8% of all new 
cancer cases (Howlader et al., 2016).  
Traditional treatment regimens for newly diagnosed MM date back to the 1960s, with 
melphalan plus prednisone as the first-line therapy along with combinations of vincristine, 
doxorubicin and dexamethasone. By the mid-1990s, HSCT became common but primarily for 
younger patients with adequate kidney function. Over the past decades, one of the major 
advances in the treatment regimen of patients with MM has been the introduction of novel 
therapies, including immune-modulating drugs (lenalidomide) and proteasome inhibitors such 
as bortezomib. Many of these novel therapies are currently recommended as initial treatment in 
newly diagnosed patients irrespective of HSCT eligibility. In the last few years, the therapeutic 
landscape of MM has improved even further with the approvals of pomalidomide, another 
immune-modulating drug, the monoclonal antibodies daratumumab and elotuzumab, as well as 


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the new-generation proteasome inhibitors carfilzomib and ixazomib. The addition of these 
novel drugs has improved both progression free (up to ~30 months) and median overall survival 
(~4.7 years) (Warren et al., 2013; San Miguel et al., 2008). There is thus agreement that eligible 
patients in second or later relapse should be considered for trial participation (Sonneveld et al. 
2017).  
1.1.3 Chimeric Antigen Receptor (CAR) T-Cells 
T cells are known to play a major role in immune surveillance and tumor eradication. Over the 
past 25 years, cancer research has been focused on developing technologies that generate, 
educate, and/or enhance T cells against tumors, and at the same time make these innovative 
treatments clinically and economically feasible. 
Adoptive Cell Therapy (ACT) is a form of immunotherapy that has demonstrated promising 
results for blood cancers and may have potential against other types of cancers as well. In 
contrast with checkpoint inhibitors, i.e. antibodies that block inhibitory molecules on T cells 
and thus unleash T-cell–mediated antitumor activity, ACT creates a productive immune 
response: through several techniques, autologous T cells are harvested, engineered to recognize 
tumor antigens, and returned to the patient to specifically target tumor cells.  
Chimeric Antigen Receptor (CAR) T-cells are engineered by combining an antibody-derived 
single-chain variable fragment (scFv) to T-cell intracellular signaling domains. These T cells 
recognize cell surface antigens in a non–MHC-restricted manner and thus do not depend on 
antigen processing and presentation. The first-generation CARs consisted of an scFv linked to 
the CD3 intracellular signaling domain. To improve persistence and proliferation of infused T 
cells, second- and third-generation CARs have been developed that incorporate the intracellular 
domains of one or multiple co-stimulatory molecules such as CD28, OX40 and 4-1BB within 
the endodomain (Houot R et al., 2015). CAR T-cells have also been designed to express 
multiple CARs that recognize several tumor antigens and stimulate production of cytokines and 
interleukins when bound to their antigen. 
Of the current trials investigating CAR T-cell therapy, 133 involve hematological malignancies 
and 78 solid tumors. Seventeen different CAR antigens are under investigation for tumors of 
the hematopoietic and lymphoid system. The most frequently targeted antigen is CD19, with 
56 ongoing and eight non-active trials. Even more antigens (22) are being investigated for the 
treatment of solid tumors. Previous trials focused on carcinoembryonic antigen targeting 
colorectal cancer, breast cancer, gastric cancer, adenocarcinoma as well as liver metastases. 
Ongoing trials target mesothelin, ErbB2/Her2, GD2 (neuroblastoma or sarcoma), or GPC3 
(hepatocellular carcinoma) (Hartmann et al., 2017). 
Promising results have been obtained in multiple myeloma with B-Cell Maturation Antigen 
(BCMA) CAR T-cells. Twenty-one (21) MM patients with  3 prior regimens including a 
proteasome inhibitor and an immunomodulatory agent or who are double refractory and have  
50% BCMA expression on MM were treated. No dose-limiting toxicity was observed and 
cytokine release syndrome (CRS) was readily manageable. Overall response rate (ORR) was 
89% with all patients receiving ≥ 150 x10^6 CAR. Minimal residual disease (MRD) negative 
status was seen in all MRD-evaluable patient samples (N=4). No patients treated with ≥ 150 
x10^6 CAR had disease progression with time from infusion between 8 and 54 weeks (Berdeja 
Jesus G et al, 2017, ASH Abstract N 740). 


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On July 1, 2014, the FDA granted ‘breakthrough therapy’ designation to CTL019, the anti-
CD19 CAR T-cell therapy developed at the University of Pennsylvania. CTL019 is the first 
personalized cellular therapy for cancer to be so designated, and 25 years after the first 
publication describing genetic redirection of T cells to a surface antigen of choice.  
Two CAR T-cell treatments are currently approved by the FDA and CHMP. Tisagenlecleucel 
(Kymriah™) is a CD19-directed genetically modified autologous T cell immunotherapy 
indicated for the treatment of patients up to 25 years of age with B-cell precursor acute 
lymphoblastic leukemia (ALL) that is refractory or in second or later relapse. The second, 
Axicabtagene ciloleucel (Yescarta™), is a CD19-directed CAR T-cell therapy for the treatment 
of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of 
systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, 
primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising 
from follicular lymphoma. 
Approval of Tisagenlecleucel was based on the ELIANA trial, a single-arm, open-label, 
multicenter, global, phase II study of CTL019 in pediatric/young adult patients with CD19+ 
R/R B-cell ALL with ≥ 5% bone marrow lymphoblasts by morphology. As of November 2016, 
88 patients were enrolled. There were 7 (8%) manufacturing failures, 9 (10%) patients were not 
infused due to death or adverse events (AEs), and 4 patients (5%) were pending infusion at the 
time of data cutoff. Following lymphodepleting chemotherapy in most patients, 68 patients 
were infused with a single dose of CTL019, with a median study follow-up of 6.4 months. 
Median age was 12 y (range, 3-23 y) and 59% of patients had prior allogeneic stem cell 
transplant (allo-SCT). Five infused patients had not reached 3 months of follow-up. Among 63 
evaluable patients, 52 (83% [95% CI, 71%-91%]) achieved CR/CRi within 3 months of 
CTL019 infusion, all of whom had minimal residual disease–negative marrow. The relapse-
free probability at 6 months after remission onset was 75% (95% CI, 57%-87%; median DOR 
not reached). The probability of survival was 89% (95% CI, 77%-94%) at 6 months and 79% 
(95% CI, 63%-89%) at 12 months. Seven patients (13% of responders) proceeded to alloSCT 
within 6 months while in remission. Cytokine release syndrome (CRS) occurred in 78% of 
patients (21% grade 3; 27% grade 4); no CRS-associated deaths occurred. The most common 
grade 3/4 nonhematologic AEs (>15%) other than CRS were hypotension (22%), hypoxia 
(18%), and increased aspartate aminotransferase (16%). The incidence of serious AEs within 8 
weeks of infusion was 69%. Fifteen percent of patients experienced grade 3 neuropsychiatric 
AEs, with no grade 4 events and no cerebral edema reported. Grade 3/4 neutropenia with high 
(> 38.3˚C) fever occurred in 60% of patients. Two patients died within 30 days of infusion (ALL 
progression, n=1; cerebral hemorrhage, n=1), and 9 patients died > 30 days after infusion (ALL 
relapse/progression, n=6; HHV-6 encephalitis, pneumonia, systemic mycosis, n=1 each). 
CTL019 expansion in vivo correlated with CRS severity, and persistence of CTL019 along with 
B-cell aplasia in peripheral blood was observed for ≥ 1 year in some responders (Buechner et 
al., 2017). 
Maude et al. conducted a Phase 2, single-cohort, 25-center, global study of tisagenlecleucel in 
pediatric and young adult patients with CD19+ relapsed or refractory B-cell ALL. The primary 
endpoint was the overall remission rate (the rate of complete remission or complete remission 
with incomplete hematologic recovery) within 3 months. Seventy-five (75) patients received an 
infusion of tisagenlecleucel and were evaluated for efficacy. The overall remission rate within 
3 months was 81%, with all patients who had a response to treatment found to be negative for 


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minimal residual disease, as assessed by flow cytometry. The rates of event-free survival and 
overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 
95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 
months. The median duration of remission was not reached. Persistence of tisagenlecleucel in 
the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were 
suspected of being related to tisagenlecleucel occurred in 73% of patients. The cytokine release 
syndrome occurred in 77% of patients, 48% of whom received tocilizumab as treatment. 
Neurologic events occurred in 40% of patients and were managed with supportive care, and no 
case of cerebral edema was reported (Maude et al., 2018). 
Approval of Axicabtagene ciloleucel was based on a multicenter, Phase 2 trial that enrolled 111 
patients with diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, or 
transformed follicular lymphoma who had refractory disease despite undergoing recommended 
prior therapy. Patients received a target dose of 2×106 anti-CD19 CAR T cells per kilogram of 
body weight after receiving a conditioning regimen of low-dose cyclophosphamide and 
fludarabine. The primary endpoint was the rate of objective response (calculated as the 
combined rates of complete response and partial response). Secondary end points included 
overall survival, safety, and biomarker assessments. Among the 111 patients who were 
enrolled, axicel was successfully manufactured for 110 (99%) and administered to 101 (91%). 
The objective response rate was 82%, and the complete response rate was 54%. With a median 
follow-up of 15.4 months, 42% of the patients continued to have a response, with 40% 
continuing to have a complete response. The overall rate of survival at 18 months was 52%. 
The most common adverse events of grade 3 or higher during treatment were neutropenia (in 
78% of the patients), anemia (in 43%), and thrombocytopenia (in 38%). Grade 3 or higher CRS 
and neurologic events occurred in 13% and 28% of the patients, respectively. Three of the 
patients died during treatment. Higher CAR T-cell levels in blood were associated with 
response (Neelapu et al., 2017). 
1.1.4 CD44v6 
Despite the remarkable results obtained by targeting CD19, widespread application of CAR 
strategy is however limited by the current lack of a CAR specific for antigens different from 
CD19. Furthermore, CD19 is not exactly a “leukemia specific” antigen. Normal B cells express 
CD19 and CD19 CAR-T cell therapy is therefore associated with the loss of CD19-positive 
normal B cells from blood and bone marrow resulting in profound hypogammaglobulinemia 
(Park et al., 2016). The impressive response rates observed in clinical trials of CD19 CAR T-
cells have in fact led to the rapid proliferation of preclinical studies testing new targets and 
methods to make CAR T-cell therapy safer and more broadly applicable to various tumor types. 
Strategies to genetically modify T cells to improve their targeting, enhance their tissue 
penetration, and control their expansion and persistence are all ways to engineer better CAR T-
cells (Maus et al., 2016). 
The hyaluronate receptor CD44 is a class I membrane glycoprotein overexpressed in 
hematologic and epithelial tumors. It plays a crucial role in the bone marrow homing of 
initiating leukemia cells and in interactions with the microenvironment. It has been shown that 
CD44 inhibition drives leukemia cells into differentiation and apoptosis by dislodging them 
from osteogenic niches (Singh et al., 2013). The isoform variant 6 (CD44v6) has recently 
emerged as one of the most promising tumor-associated antigens (TAA) for AML. CD44v6 is 
expressed at variable levels in 16 of 25 (64%) AML cases belonging to different French-


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American-British subtypes and World Health Organization categories and in 13 of 15 (87%) 
MM cases at different stages according to the Durie-Salmon classification or the International 
Staging System (Casucci et al., 2013). Preclinical studies have shown that CD44v6-silenced 
THP-1 (AML) and MM.1S (MM) cells are severely impaired in their capacity to engraft in 
immune-compromised mice and, similarly, CD44v6-silenced primary leukemic blasts fail to 
initiate leukemia in vivo, providing further rationale for targeting the CD44v6 antigen in AML 
and MM since the potential generation of antigen lost variants is circumvented by the reduced 
growth of CD44v6 negative tumor cells. 
1.1.5 EURE-CART Project 
This clinical study protocol is Working Package 4 of grant N. 733297 awarded by the European 
Commission for the action entitled ‘EURopean Endeavour for Chimeric Antigen Receptor 
Therapies — EURE-CART.  
The nine Parties described in Annex 1 formed a consortium and signed a grant agreement with 
the European Commission, under the coordination of MolMed, which acts also as a Sponsor of 
this clinical study. 
 Drug description 
Generation of the medicinal product (MLM-CAR44.1 T cells) 
The medicinal product (MLM-CAR44.1 T cells) is defined as frozen T lymphocytes genetically 
modified to express CAR CD44v6 by the retroviral vector CAR CD44v6ΔNL encoding the 
CAR CD44v6 and HSV-TK Mut2 genes in the final formulation and container closure system, 
ready for intended medical use. It is a patient-specific product prepared starting from a 
lymphocyte apheresis of the patient.  
The retroviral vector CAR CD44v6ΔNL is generated from the SFCMM3 Mut2 vector currently 
used for manufacturing Zalmoxis (i.e. encoding the HSV-TK Mut2 suicide gene and the 
LNGFR marker gene) by substituting the marker gene with CAR CD44v6. 
In its final version, the CAR CD44v6ΔNL retroviral vector carries the HSV-TK Mut2 gene 
under the transcriptional control of the viral 5’LTR promoter and an internal expression cassette 
with the SV40 promoter driving the CAR CD44v6.  
The CAR CD44v6 includes: 
 the single-chain variable fragment (scFv) of the humanized mAb BIWA-8 (Verel et 
al., 2002) specific for the CD44v6 antigen;  
 a spacer formed by the extracellular domain of the human low-affinity nerve growth 
factor receptor (LNGFr) that allows the selection of cells to be infused in patients 
and their monitoring using specific antibodies (Casucci et al., 2018); 
 the transmembrane and intracellular domain of the human costimulatory molecule 
CD28;  
 the intracellular signaling domain of the human CD3ζ chain. 
The HSV-TK Mut2 suicide gene encodes a mutated form of the thymidine kinase enzyme of 
Herpes Simplex Virus I and provides a safety valve in case of toxicity. Indeed, the derived 
enzyme is functional and used both in vitro and in vivo to selectively eliminate the transduced 


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cells in presence of Ganciclovir (GCV). The HSV-TK Mut2 variant was generated by site-
directed mutagenesis with the introduction of a silent T to C transition at a splicing donor site 
of the original HSV-TK. The mutation has been proven to avoid the generation of GCV-
resistant HSV-TK spliced forms. 
To generate the stable packaging cell line producing the CAR CD44v6ΔNL retroviral vector, 
the LTK-S44v6ΔNL plasmid was used to transfect the ecotropic GP+E86 packaging cells line 
(ATCC n° CRL-9642). The supernatant from the transient transfection was harvested and used 
to infect the PG13 packaging cell line (ATCC n° CRL-10686) expressing the Gibbon Ape 
Leukemia virus envelope protein (Galv). The integration of the construct in the packaging cell 
line DNA allows the production of retroviral vectors able to infect a broad range of mammalian 
cells, including human cells. The PG13 bulk cell population was selected, following 
transduction, based on expression of CD44v6 CAR using antibodies specific for the LNGFR 
spacer conjugated to magnetic beads. The resulting transduced cells were then plated in limited 
dilution conditions to obtain producer cell clones. The clones were tested for expression of 
CD44v6 CAR, good growth capacity in culture, efficiency of transduction of T lymphocytes, 
stability, absence of contaminating adventitious viruses and of replication-competent forms. 
The producer clone PG13-187#68 was chosen for further development. 
The MLM-CAR44.1 T-cell manufacturing process essentially consists in: i) stimulation of 
peripheral blood mononuclear cells isolated from patients with TransAct, a polymeric 
nanomatrix conjugated to anti-CD3 and anti-CD28 mAbs, IL15 and IL7; ii) transduction of the 
stimulated cells with the CAR CD44v6ΔNL retroviral vector using RetroNectin®; iii) 
immunoselection of the transduced cells using CD271 Microbeads. Selected cells are then 
further expanded before the final formulation in frozen medium. 
Of note, the overall yield of the MLM-CAR44.1 T-cells manufacturing process carried out with 
AML starting material is quite variable and lower to what observed with peripheral blood 
mononuclear cells (PBMC) from healthy donors due to the lower CD3+ percentages and the 
presence of variable degrees of leukemic cells. However, the expansion of CD3+ cells during 
the culture period is good, indicating that if a proper number of CD3+ cells is present in the 
patient apheresis/starting material, it should be feasible to reach the desired final dose of MLM-
CAR44.1 T-cells. 
The entire manufacturing process lasts 10 days and is performed in an open system, except for 
cell washing and CD271 selection at day 8, which employ the CliniMACS Prodigy instrument.  
Overall, the transduced cells are positive for the CD45 leukocyte marker, and for CD3+. No 
relevant proportions of monocytes (CD14+), B cells (CD19+) and NK cells (CD3- CD16+ and 
CD56+) are found. T-lymphocyte subpopulations (CD4+ and CD8+) are represented in variable 
degrees among different patients. 
 
 Drug activity 
The expression of CAR CD44v6 allows the transduced T-cells to acquire a potent and specific 
reactivity against tumor cells expressing the CD44v6 antigen, including primary AML and MM 
cells.  


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Moreover, the expression of the CAR CD44v6 on the cell membrane allows for the 
immunoselection of genetically modified cells using mAb conjugated to magnetic beads that 
recognize the LNGFR spacer. The expression of the LNGFR protein can be used in vivo as a 
marker of genetically modified cells once infused into the patients and allows the assessment 
of drug product persistence in the human body following treatment, possible cell population 
expansion or reduction and characterization in terms of lymphocyte subtype and state of 
activation.  
HSV-TK Mut2 makes the transduced cell sensitive to GCV. Following its administration, GCV 
is first monophosphorylated by the HSV thymidine kinase enzyme expressed in the genetically 
modified cells and then transformed to the triphosphate form by other cellular kinases. This 
active, triphosphate form of GCV induces the blockage of the synthesis of new DNA strands 
during mitosis, and hence provokes the death of proliferating cells. 
1.3.1 Non-clinical studies 
Pre-clinical studies: efficacy 
The original CD44v6 CAR construct (CH2CH3-spaced CAR) was developed at the research 
laboratories of the San Raffaele Institute. When co-cultured with an excess of target cells, 
CD44v6.CAR28z T cells efficiently eliminated CD44v6+, but not CD44v6–, primary leukemic 
blasts and malignant plasma cells. The antitumor activity of these CAR T cells was then 
assessed in vivo in NOD/SCID/IL2Rnull (NSG) mice previously infused with primary leukemic 
blasts and then treated with a single infusion of CD44v6.CAR28z or control CTR.CAR28z T 
cells from the same mouse. Although control mice had increasing percentages of circulating 
leukemic blasts over time, mice receiving CD44v6.CAR28z T cells had progressive 
disappearance of leukemia and, at sacrifice, were completely cleared of tumor cells in the bone 
marrow. 
The original CD44v6 CH2CH3-spaced CAR however mediated suboptimal long-term 
antitumor activity in vivo when challenged against high tumor burdens due to undesired 
interaction with Fc receptor (FcR)-bearing myeloid cells and premature clearance. To develop 
a more effective CAR construct that could enable enrichment and tracking of CD44v6 CAR T 
cells, the CH2CH3 spacer was replaced with the extracellular domain from the human low 
affinity nerve growth factor receptor (NL) currently used as cell surface marker in Zalmoxis 
cell therapy. In co-culture experiments with tumor cells expressing CD44v6 and/or FcR, the 
new construct killed CD44v6+ THP-1 myeloid leukemia cells and MM.1S myeloma cells but 
spared CD44v6− BV173 lymphoid leukemia cells. They also failed to kill CD44v6−/FcR+ HL60 
myeloid leukemia cells, indicating efficient abrogation of FcR recognition. Importantly, 
maintenance of potent antitumor reactivity in these CD44v6ΔNL CAR T cells was confirmed 
by efficient and specific killing of both primary AML blasts and malignant plasma cells 
expressing CD44v6. Finally, antitumor activity was tested in vivo in the THP-1 leukemia and 
MM.1S myeloma models. Treated mice benefited from substantial antitumor effects, as 
indicated by normalization of THP-1 cell infiltrated liver weight. Moreover, similar results were 
obtained when testing against myeloma. 
A GLP-like study was then conducted to determine the antitumor efficacy of CAR T cells 
transduced with CD44v6ΔNL CAR and HSV-TK-encoding retrovirus according to the final 
drug product manufacturing process (44v6.28z cells). 44v6.28z cells were compared to 
CD19ΔNL CAR T cells identically cultured and manipulated (19.28z cells). In co-culture 


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experiments with tumor cells, 44v6.28z cells were able to efficiently kill MM.1S cells, 
proliferate and produce inflammatory cytokines. THP-1 cells were also targeted, although with 
inferior intensity due to lower CD44v6 expression levels. Moreover, CD44v6neg BV-173 cells 
were completely spared. 
The in vivo functionality of 44v6.28z cells was tested in NSG mice engrafted with tumor cells. 
MM.1S myeloma cells and THP-1 leukaemia cells were modified to express a secreted 
luciferase, which has the great advantage of being released by the cells and is therefore 
detectable in blood allowing monitoring of tumor progression in mice. In the myeloma model, 
treatment did not cause toxicity per se, as indicated by the general well-being of mice and the 
lack of consistent weight loss. 44v6.28z cells significantly inhibited tumor progression in mice, 
as indicated by bioluminescence signals measured in blood. The antitumor effect of 44v6.28z 
cells was confirmed at sacrifice, when no or very low percentages of tumor cells were found 
both in the bone marrow and in the spleen. Notably, antitumor activity was paralleled by CAR 
T cell expansion and contraction. 44v6.28z cells were predominantly CD4+ and, in accordance 
with antigen-specific recognition, were strongly activated, as indicated by transient increases 
of HLA-DR. Importantly, the expression of PD1 was only transiently up-regulated at the peak 
of T cell activation and then returned to basal levels, indicating no CAR T cell exhaustion and 
thus long-term antitumor potential. 
In the leukemia model, treatment with 44v6.28z cells was well tolerated and able to 
significantly inhibit tumor progression, as indicated by both luciferase levels and liver weight 
at sacrifice. CAR-T cells expanded in peripheral blood, were predominantly CD4+ and were 
activated upon antigen encounter. Also in this setting, 44v6.28z cells were detectable at 
sacrifice both in the bone marrow and spleen, still included a proportion of early differentiated 
cells and did not express exhaustion and senescence markers. 
Full details of preclinical studies are available in the Investigator’s brochure. 
Pre-clinical studies: safety 
In clinical trials, tumor responses by CAR-redirected T cells were often associated with 
toxicities deriving from off-tumor expression of the target. Cases of fatal lung toxicity and the 
frequent observation of prolonged B-cell depletion were observed using HER2 and CD19 CAR 
T-cells, respectively. Despite promising activity against epithelial tumors, the administration of 
the CD44v6-specific mAb (bivatuzumab) used for deriving our CAR scFv showed reversible 
myelosuppression and mucositis when conjugated with radioisotopes (Borjesson et al., 2003), 
and showed skin toxicity, including a fatal case, when conjugated with the potent cytotoxic 
drug mertansine (Tijink et al., 2006). 
In accordance with CD44v6 expression on monocytes and keratinocytes, but not on HSCs and 
progenitor cells, the infusion of CD44v6 CAR T cells in human hematochimeric mice caused 
selective and reversible monocytopenia, indicating preservation of the HSC pool. Indeed, after 
in vivo exhaustion of CD44v6-targeted T cells, NSG-3G mice reconstituted monocytes de novo 
(Casucci et al., 2013). 
Keratinocytes appear to be resistant to CD44v6 CAR T activity in vitro, whereas they are killed 
by EGFR CAR T-cells. To verify in vivo the absence of activity against skin-resident 
keratinocytes, we developed a human-skin xenograft mouse model. Full-thickness human skin 
fragments were implanted subcutaneously into immunodeficient NSG mice. After healing of 


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the skin graft, mice were treated with either vehicle, unrelated CD19 CAR T-cells (negative 
control), EGFR CAR T-cells (positive control) or CD44v6 CAR T-cells (test item). Three days 
after CAR T-cell infusion, no differences were found in the number of circulating T cells 
between the different groups, while we observed an increased percentage of cells expressing 
HLA-DR in EGFR CAR T-cells as compared to CD44v6 CAR T-cells, indicating a more 
activated state. Two weeks after CAR T-cell infusion, skin xenografts were recovered and 
analyzed by immunohistochemistry for the presence of infiltrating human T cells. Mice that 
were infused with EGFR CAR T-cells showed the highest CD3+ cell infiltration in the derma 
portion of the skin xenograft, while mice infused with CD44v6 CAR T-cells showed only low 
CD3+ cell infiltration, similarly to control mice receiving CD19 CAR T-cells. At the dermal-
epidermal junction of the skin, we observed a similar trend without reaching statistical 
significance. These results were confirmed by using a skin explant model in which human skin 
biopsies were directly co-cultured with CAR T cells for 3 days before being analyzed by 
immunohistochemistry. Also in this model, minimal skin infiltration by CD44v6NL CAR T 
cells was reported. 
The TK/ganciclovir system incorporated into the CAR construct was designed to address 
potential CAR T-cell toxicities, and its efficacy was confirmed in animal models. 44v6.28z 
cells generated from two different healthy donors expanded in the peripheral blood of mice 
receiving saline, while they remained nearly undetectable in mice treated with ganciclovir 
(GCV). These results were confirmed at sacrifice as no 44v6.28z cells were identified in the 
bone marrow of mice treated with GCV, while they were present in control mice. Besides being 
effective, the suicide system proved to be specific since no significant differences were 
observed in mice infused with untransduced T cells and treated with either saline or GCV. 
All together, these results and the presence of a suicide gene in the transduced cells, strongly 
suggest that therapeutic doses of CD44v6 CAR T-cells are likely associated with acceptable 
and/or reversible toxicities.  
Full details of preclinical safety studies are available in the Investigator’s brochure. 
1.3.2 Clinical studies 
Not Applicable 
 Rationale – risks and benefits 
1.4.1 Possible benefits of CAR-T cell therapy 
Even in this epoch of great drug development, there is urgent need to investigate how new 
therapies and interventions may further benefit patients with MM and AML. In this context, 
CAR T-cells represent an exciting frontier in cancer therapy: antitumor T cells with high-avidity 
recognition of tumor antigens can be expanded in vitro in large numbers, genetically 
engineered, and/or activated ex vivo to acquire antitumor functions. In addition, the host can be 
manipulated before cell transfer to eliminate suppressor cells and promote in vivo expansion of 
transferred lymphocytes, which can then function as “living drugs” that induce long-term 
protection (Houot et al., 2015). 
CAR T-cell therapy is entering a new era, transitioning from an experimental approach being 
tested in a handful of centers to a more mainstream and broadly investigated therapeutic 


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platform with significant efforts directed towards commercial translation. Ongoing innovations 
into the design and application of CAR T-cells are aimed at improving antitumor potency and, 
at the same time, ensuring safety. The remarkable clinical efficacy demonstrated with CD19 
CARs has been achieved at multiple institutions, each evaluating their own CAR T-cell 
platforms and trial designs. The principles that have arisen from this wealth of clinical 
experience has helped shape the parameters key to achieving therapeutic success, as well as the 
management of potential toxicity (Brown et al., 2016). 
The results of preclinical studies have demonstrated that CD44v6ΔNL CAR T cells efficiently 
and consistently inhibit tumor growth in both MM and AML models. The correlation between 
antitumor activity and level of antigen expression suggests that CD44v6ΔNL CAR T cells have 
highly specific targeting properties that should favor low toxicity in vivo. Moreover, their 
phenotypes (i.e. significant proportion of early differentiated T cells and few exhausted cells) 
may potentially provide long-term antitumor effects in patients. 
Given the refractory and relapsing nature of AML and MM, the high levels of CD44v6 surface 
expression on tumor cells and the results of preclinical studies, the aim of the present study is 
to take CAR T-cell therapy one step further by investigating the treatment of these diseases 
with CD44v6 CAR T-cells (MLM-CAR44.1 T-cells). The purpose of this first-in-man, Phase 
I-IIa study is to evaluate the safety and antitumor activity of MLM-CAR44.1 T-cells in patients 
with AML and MM. 
Considering the “first in human” nature of this clinical study, the Bayesian Optimal Interval 
Design (BOIN) (Liu and Yuan, 2015) has been chosen to minimize any risks of exposure to the 
novel MLM-CAR44.1 T-cells during dose escalation. The BOIN design is a novel Bayesian 
dose-finding method that minimizes the decision errors of dose assignment (for example the 
chance of exposing patients to overly toxic doses) and thus ensures patient ethics. This design 
has performance superior to the traditional 3+3 design (ref Liu and Yuan, 2015), but shares 
with it the fact that it can be easily implemented, differently from the well-known continual 
reassessment method (CRM), the gold standard of Phase 1 dose finding. The BOIN design 
yields an average comparable performance compared to CRM in selecting the MTD but has a 
lower risk of assigning patients to sub-therapeutic or overly toxic doses. 
1.4.2 Possible risks of CAR T-cell Therapy 
The adverse effects of CAR T-cell therapy can vary from mild constitutional syndrome (flu-
like syndrome, malaise, fever, etc.) to life threatening situations requiring intensive care. 
Toxicities can be summarized as follows (Hartman et al., 2017): 
Cytokine-release syndrome (CRS): A systemic inflammatory response resulting in non-
infectious fever with elevated levels of inflammatory cytokines such as interleukin-6 and 
interferon-γ. 
On-target/off-tumor toxicity: Adverse effects caused by the killing of healthy tissue by CAR T-
cells due to target antigen expression outside tumor tissue. 
Off-target toxicity: Side effects in CAR T-cell-treated patients due to cross-reactivity of the 
engineered antigen binding domain with a non-related surface protein. 
Neurotoxicity: Presence of neurocognitive deficits. 


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Allergic reactions and Tumor Lysis Syndrome (TLS), the latter a group of metabolic 
complications that can occur due to the breakdown of dying cells. 
Infectious diseases caused by opportunistic agents in immune-compromised patients after 
administration of chemotherapy followed by CAR T-cells. 
Insertional oncogenesis, transformation of a T-cell clone and clonal expansion. 
1.4.2.1 Cytokine release syndrome 
Cytokine release syndrome (CRS) is a novel critical illness syndrome characterized by a series 
of inflammatory symptoms resulting from cytokine elevations associated with T-cell 
engagement and proliferation. In most patients, CRS symptoms are mild and flulike, with fever 
and myalgia. However, some patients have experienced a severe inflammatory syndrome that 
includes vascular leakage, hypotension, pulmonary edema, and coagulopathy, resulting in 
multiorgan system failure and in death, the latter related to treatment with blinatumomab. In 
the study by Maude et al., severe cytokine-release started a median of one day after infusion, 
whereas non-severe CRS started 4 days later (Maude et al., 2014). 
A widely referenced mechanism for grading CRS due to T-cell therapies is the one proposed 
by Neelapu et al. Grade 1 symptoms include fever and Grade 1 organ toxicity and require only 
symptomatic management. Grade 2 symptoms respond to moderate intervention and include 
oxygen requirement <40%, grade 2 organ toxicity, or hypotension responding to IV fluids or 
low doses of one vasopressor (e.g., < 20 μg/min of norepinephrine). Grade 3 CRS includes an 
oxygen requirement ≥ 40%, hypotension requiring high-dose or multiple vasopressors, grade 3 
or 4 transaminitis, and grade 3 organ toxicity at other sites. Grade 4 CRS is defined as the 
presence of life-threatening symptoms requiring ventilator support or grade 4 organ toxicity 
other than transaminitis (Neelapu et al., 2017). Grade 4 CRS will be considered a Dose Limiting 
Toxicity (DLT) for the definition of Maximum Tolerated Dose (MTD) in Phase I of the present 
study. The same applies to Grade 3 or higher CRS not responsive to therapy with steroids and 
or tocilizumab/siltuximab within 24 hours. 
A strategy for managing CRS is to target cytokines directly. The cytokines most significantly 
elevated in CRS associated with CD19 CAR T-cells and blinatumomab were IL-10, IL-6, and 
IFN-γ. IL-10, primarily produced by monocytes/macrophages, regulates both innate and cell-
mediated immunity by inhibiting activated macrophages. IL-10 can also be produced by mast 
cells, B cells, regulatory T cells, and helper T cells (TH2), but not commonly by cytotoxic T 
lymphocytes. As a negative regulator, IL-10 may not be an ideal cytokine to target in CRS. 
Conversely, IL-6 is an inflammatory cytokine involved in many processes within the immune 
system, such as neutrophil trafficking, acute phase response, angiogenesis, B-cell 
differentiation, and autoantibody production. IL-6 is produced by monocytes/macrophages, 
dendritic cells, T cells, fibroblasts, keratinocytes, endothelial cells, adipocytes, myocytes, 
mesangial cells, and osteoblasts. Interferon-γ is also an inflammatory cytokine and is involved 
in macrophage activation, TH1 differentiation, major histocompatibility complex class 1 
induction, and B-cell isotype switching. Interferon-γ is produced by cytotoxic T cells, helper T 
cells (TH1), and natural killer cells. As an effector cytokine released by activated cytotoxic T 
cells after engagement, IFN-γ elevations are both anticipated and likely required for efficacy, 
making it a potentially undesirable target for toxicity management (Maude et al., 2014). 


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Based on these considerations, targeting IL-6 represents an effective strategy for reducing CRS 
toxicity without compromising the clinical efficacy of T-cell therapies. IL-6 blockade by 
tocilizumab determined rapid, dramatic reversal of life-threatening CRS in patients treated with 
CD19 CAR T-cells or blinatumomab (Grupp et al., 2013; Maude et al., 2014). In patients who 
responded to tocilizumab, fever and hypotension often resolved within a few hours, and 
vasopressors and other supportive care measures were weaned quickly thereafter. In some 
cases, symptoms may however not completely resolve, and continued aggressive support may 
be necessary for several days, along with the administration of a second dose of tocilizumab 
and/or a second immunosuppressive agent such as corticosteroids (Lee et al., 2014). 
Tocilizumab is a recombinant humanized monoclonal antibody that blocks IL-6 from binding 
to its receptor and is approved in the EU for the treatment of moderate to severe active 
rheumatoid arthritis in adult patients and for the treatment of juvenile idiopathic arthritis in 
children as young as 2 years. On 30 August 2017, the FDA granted Priority Review and Orphan 
Drug Designation to tocilizumab intravenous injection for the treatment of CAR T cell-induced 
severe or life-threatening CRS in patients two years of age and older. The approval was based 
on a retrospective analysis of pooled outcome data from clinical trials of CAR T-cell therapies 
for blood cancers (Grupp et al., 2016). The study population included 45 pediatric and adult 
patients treated with tocilizumab, with or without additional high-dose corticosteroids, for 
severe or life-threatening CRS. Thirty-one patients (69%; 95% CI: 53%–82%) achieved a 
response, defined as resolution of CRS within 14 days of the first dose of tocilizumab, no more 
than two doses of tocilizumab were needed, and no drugs other than tocilizumab and 
corticosteroids were used for treatment. No adverse reactions related to tocilizumab were 
reported. A second study confirmed resolution of CRS within 14 days using an independent 
cohort that included 15 patients with CAR T cell-induced CRS. 
Grade 4 CRS or Grade 3 CRS not responsive to steroids and/or tocilizumab within 24 hours 
may set off suicide gene activation. 
1.4.2.2 On-target/off tumor recognition 
The ideal target antigen is restricted to the tumor cell. Unfortunately, most targets of CAR T-
cells have shared expression on normal tissues and some degree of “on-target/off-tumor” 
toxicity occurs through engagement of target antigen on nonpathogenic tissues. One of the 
earliest trials utilizing a carboxyanhydrase-IX-specific CAR T-cell for renal cell carcinoma 
resulted in the development of cholestasis due to expression of carboxyanhydrase-IX on bile 
duct epithelium (Lamers et al., 2013). In the setting of CD19-specific CAR T-cells, the targeting 
of normal B cells results in B-cell aplasia, which may require intermittent infusion of pooled 
immunoglobulin as prophylaxis from infectious complications (Bonifant et al., 2016). A fatal 
example of “on-target/off-tumor” recognition is that of a patient treated with CAR T-cells 
specific for the cancer-associated antigen HER-2/neu. The patient developed rapid respiratory 
failure, multi-organ dysfunction, and subsequent death attributed to reactivity against 
pulmonary tissue expression of HER-2/neu (Morgan et al., 2010). However, this unforeseen 
toxicity was potentially provoked by the substantial dose of infused CAR T cells (1 × 1010 CAR 
T cells) as subsequent studies utilizing a different HER2/neu-specific CAR (without prior 
conditioning chemotherapy) have proven safe at significantly lower CAR T-cell doses (Ahmed 
et al., 2015). 


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CD44v6 has a low level of expression on normal cells, including activated T cells, monocytes, 
and keratinocytes. Preclinical studies have shown that on-target off-tumor toxicities associated 
with CD44v6 CAR T-cells are acceptable and/or reversible (see Section 1.3.1) Nevertheless, 
given the potential for off-tumor toxicity of CD44v6 CAR T-cells, patients will be closely 
monitored for skin toxicities and monocyte levels. Moreover, patients with multiple antibiotic 
resistant diseases will not be eligible for this study. A DLT for Phase I of this study is the 
presence of NCI-CTC grade 3 or higher erythroderma (generalized exfoliative dermatitis), 
Stevens-Johnson Syndrome, Toxic Epidermal Necrolysis (TEN), and any other grade 3 or 
higher skin toxicity suspected of being related to CAR T-cell treatment. 
Based on the clinical judgment of the Investigator, and in the presence of the following off-
tumor skin toxicities, ganciclovir may be administered to set off suicide gene activation: 
 Grade 3 erythroderma not responsive to steroids (methylprednisolone 2mg/kg for 72 
hours or rapidly evolving) 
 Grade 2 or higher bullous dermatitis 
 Any grade Stevens Johnson Syndrome 
 Any evidence for Toxic Epidermal Necrolysis (TEN) 
1.4.2.3 Off-target antigen recognition 
Most CAR T-cells recognize antigen through scFv derived from monoclonal antibodies, some 
of which may have a proven safety record in clinical use while others may not. Organ damage 
could hypothetically occur when CAR T-cells cross-react with antigens expressed on normal 
tissue that are similar to the target antigen expressed by the malignancy. This toxicity has not 
been documented in clinical trials of CARs but has been observed in clinical trials of T cells 
genetically modified to express T-cell receptors (Brudno et al., 2016). 
Predicting off-target antigen recognition and subsequent toxicities is difficult in first-in-man 
studies. To this end, patients will be diligently monitored to recognize any potential toxicity 
promptly. 
1.4.2.4 Neurotoxicity 
Neurologic toxicities including confusion, delirium, expressive aphasia, obtundation, 
myoclonus, and seizure have been reported in patients receiving CD19-specific CAR T-cells 
(Maude et al., 2014, Davila et al., 2014, Lee et al., 2015). 
The pathophysiology of these neurologic side effects is unknown although it is plausible that 
elevated cytokine levels are partly responsible. Conversely, direct CAR T-cell toxicity on the 
central nervous system is possible but has not been demonstrated. Neurological events may 
occur at different times than CRS or in the absence of CRS toxicities (Maude et al., 2014), 
suggesting that neurologic toxicity might have a different mechanism than other toxicities such 
as fever and hypotension. 
In most instances, neurologic events are self-limiting, and there are no definitive guidelines 
regarding best management of these events. It is unclear if tocilizumab has any beneficial effect. 
Because tocilizumab is a monoclonal antibody, its size makes efficient Blood-Brain Barrier 
(BBB) penetration unlikely. The smaller IL-6 molecule is known to cross the BBB and has been 
shown to cause neurologic defects. Saturation of IL-6 receptors following systemic tocilizumab 


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administration may increase serum IL-6 levels, theoretically leading to an increase in CSF IL-
6 levels that might worsen neurologic toxicity. As for other groups (Lee et al., 2014), the 
Transplantation and Immunology Branch of the US National Cancer Institute treats severe 
neurologic toxicities with systemic corticosteroids rather than tocilizumab as the first-line 
agent. (Brudno et al., 2016). 
Grade 3 or higher neurotoxicity will be considered a DLT in this study and may trigger suicide 
gene activation. 
1.4.2.5 Allergic reactions and Tumor Lysis Syndrome (TLS) 
Allergic reactions to CAR T-cells have been reported. A patient with pleural mesothelioma 
received multiple infusions of autologous T cells transduced with an antimesothelin CAR. 
Although the patient tolerated the first two cell infusions well, he experienced anaphylaxis and 
cardiac arrest following completion of the third infusion, with dramatically elevated serum 
tryptase levels. The patient received cardiopulmonary resuscitation and recovered (Maus et al., 
2013). 
Although chemotherapy may have caused TLS in some cases, the infusion of CAR T cells in 
the absence of prior conditioning chemotherapy has led to TLS (Kochenderfer et al., 2013; 
Grupp et al., 2013).  
Given the growing interest in CAR T-cell therapies, the CAR-T-cell therapy-associated 
Toxicity (CARTOX) Working Group (comprising investigators from multiple institutions and 
medical disciplines who have experience in treating patients with various CAR-T-cell therapy 
products) has published a series of recommendations for monitoring, grading, and managing 
the acute toxicities that can occur in patients treated with CAR T-cell therapy (Neelapu et al, 
2017). These recommendations are aimed at reducing CAR T-cell associated mortality and 
morbidity by optimizing intensive monitoring, accurate grading, and prompt management of 
these toxicities with aggressive supportive care, anti-IL-6 therapy, and/or corticosteroids for 
severe cases. 
1.4.2.6 Infectious diseases 
Patients on CAR T-cell clinical trials frequently become neutropenic and lymphopenic after 
administration of chemotherapy followed by CAR T-cells, predisposing them to opportunistic 
infection. In this setting, the fevers, tachycardia, and hypotension associated with CRS can be 
difficult to differentiate from sepsis. In an early report, a patient with chronic lymphocytic 
leukemia who received chemotherapy and anti-CD19 CAR T-cells died with fever, 
hypotension, and renal failure. It was later found that this patient had elevated serum levels of 
inflammatory cytokines before CAR T-cell infusion, suggesting that the patient had a prior 
infection (Brentjens et al., 2010). Bacteremia, Salmonella, urinary tract infections, and viral 
infections such as influenza, respiratory syncytial virus, and herpes zoster virus, have also 
occurred following CAR T-cell infusion. 
Given their predisposition to infections, patients with multiple antibiotic resistant diseases will 
not be eligible for this study. 
Suicide gene activation may be considered in case of grade 3-4 potentially life-threatening 
infections. 


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1.4.2.7 Insertional oncogenesis 
Insertion of a transgene into differentiated T cells carries the risk of induced malignant 
transformation. However, no adverse or toxic events related to the gene transfer procedure have 
been reported to date. Accordingly, no genotoxic effect of integrating vectors, nor clonal 
dominance of gene modified T-cells has been observed (Recchia et al., 2006; Lupo Stanghellini 
et al., 2014). 
1.4.3 Suicide gene 
As described above, CAR T-cell therapy may lead to several types of toxicity due to the 
inability to control T-cell activity and some tumor-associated antigens that are presented by 
both tumor and healthy tissue. A safe and efficient means of resolving these adverse effects is 
the incorporation of suicide genes into the CAR T-cells. With this approach, T cells are 
engineered to express signaling pathways that cause the T cell to destroy itself after a defined 
number of cell divisions. 
Several “suicide-gene” strategies that allow selective destruction of administered T-cells on 
demand have been developed. A suicide gene codes for a protein able to convert, at a cellular 
level, a non-toxic prodrug into a toxic product. Expression of the herpes simplex virus 
thymidine kinase (HSV-TK) renders modified cells susceptible to treatment with the acyclic 
nucleoside analog ganciclovir (GCV). Once expressed, HSV-TK catalyzes phosphorylation of 
GCV resulting in competitive inhibition of guanosine incorporation with subsequent disruption 
of DNA polymerization and synthesis (Greco et al., 2015). The killing mechanism of the 
TK/GCV suicide gene is unique in that GCV kills only proliferating engineered cells, allowing 
for resting lymphocytes to be spared for subsequent therapy and immune reconstitution 
(Oliveira et al., 2012). GCV administration in most of the cases eliminates more than 90% of 
circulating TK cells (Ciceri et al., 2009). Of note, transduced T-cells can be easily tracked in 
patients thanks to the presence of the LNGFR spacer in the MLM-CAR44.1 T-cell, thus 
providing a unique tool to study the fate of the CAR T-cells in vivo (Bonini et al., 2003). 
Since suicide gene therapy for allogenic HSCT has demonstrated the potential to safely balance 
graft versus tumor (GvT) and graft versus host disease (GvHD) (Greco et al., 2015), the 
implementation of a suicide gene in MLM-CAR44.1 T-cells should help to mitigate their risks 
while preserving their therapeutic effects (Casucci et al., 2013). Although CAR T-cells are in 
general well tolerated, their broader use requires having solid strategies to treat on-target, off-
tumor effects and cytokine storms (Hoyos et al., 2010). Applying suicide gene modification to 
MLM-CAR44.1 T-cells may greatly increase their safety profile and facilitate their clinical 
development. 
In conclusion, the risk to subjects in this trial should be minimized by eligibility criteria, pre-
infusion criteria, a CRS treatment algorithm, study procedures and close clinical monitoring. 
Based on the high response rates and lasting remissions of CTL019 therapy, the potential 
benefit of treatment with MLM-CAR44.1 T-cells in the target patient population outweighs its 
potential risks. 
 
 


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2 
OBJECTIVES 
 Phase I objectives 
2.1.1 Primary objectives 
1. To determine the maximum tolerated dose (MTD) and recommended Phase II dose of 
MLM-CAR44.1 T-cells in patients with relapsed/refractory acute myeloid leukemia 
(AML) or multiple myeloma (MM) expressing CD44v6.  
The MTD will be established through a BOIN study design and the following dose-
limiting toxicities (DLT) occurring within 30 days following CAR T-cell infusion: 
a. Grade 4 CRS 
b. Grade 3 or higher CRS not responsive to therapy with steroids and/or 
tocilizumab/siltuximab within 24 hours 
c. NCI-CTC grade 3 or higher toxicity at least possibly related to treatment with 
MLM-CAR44.1 T-cells excluding hematological toxicities 
d. Skin toxicities:  
o 
NCI-CTC grade 3 or higher erythroderma (generalized exfoliative 
dermatitis) 
o 
Stevens-Johnson Syndrome 
o 
Toxic Epidermal Necrolysis (TEN) 
o Any other grade 3 or higher skin toxicity histologically confirmed as being 
related to CAR T-cell treatment 
e. Grade 3 or higher neurotoxicity 
2. To evaluate the overall safety of treatment with MLM-CAR44.1 T-cells. 
Overall safety will be evaluated by analyzing the type, frequency and severity of adverse 
events (AE) and by monitoring for systemic reactions (fever, tachycardia, nausea and 
vomiting, joint pain, skin rash) for 30 days following CAR T-cell infusion. 
3. The absence of replication-competent retrovirus (RCR) will be monitored by DNA PCR 
3, 6, 12 and 24 months after infusion and then yearly as defined in the long-term follow-
up. 
2.1.2 Secondary objectives 
1. To evaluate hematologic response to MLM-CAR44.1 T-cells in AML and MM. 
The endpoints for assessing hematologic response in Phase I are defined as follows: 
a. AML: complete remission (CR), incomplete response (CRi) and partial response 
(PR) according to European LeukemiaNet (ELN) criteria, 1 and 2 months following 
infusion. 
b. MM: overall response rate (ORR): stringent complete response (sCR), CR, very 
good partial response (VGPR) and partial response (PR) according to International 
Myeloma Working Group (IMWG) criteria, 1 and 3 months following infusion. 
2. To characterize the in vivo pharmacokinetic profile (engraftment, persistence, 
trafficking) of MLM-CAR44.1 T-cells by evaluating levels of circulating MLM-
CAR44.1 T-cells by flow cytometry. 


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3. To assess suicide gene activation and elimination of transduced cells through 
administration of ganciclovir in case of CRS and other MLM-CAR44.1 T-cell related 
toxicities, by evaluating levels of circulating MLM-CAR44.1 T-cells by flow 
cytometry. 
4. To conduct an ancillary study including but not limited to the characterization of the 
cellular composition of circulating LNGFR+ (CAR T-cells) and LNGFR- cells, and the 
monitoring of the patient’s immune status through analyses of peripheral blood 
mononuclear cells (PBMC). 
5. To set-up a validation process among the participating centers for the CD44v6 marking 
of tumor cells (AML and MM).  
 
 Phase IIa objectives 
2.2.1 Primary objective 
To evaluate hematologic response to MLM-CAR44.1 T-cells at 2 and 3 months after infusion 
in AML and MM, respectively 
The primary endpoint is disease response, defined as: 
AML: CR, CRi and PR rates as per ELN criteria, 2 months after MLM-CAR44.1 T-cell 
infusion. 
MM: ORR: (sCR, CR, VGPR and PR) as per IMWG criteria, 3 months after T-cell 
infusion. 
2.2.2 Secondary objectives 
1. To evaluate the antitumor activity of MLM-CAR44.1 T-cells 
The secondary endpoints for assessing antitumor activity are as follows: 
 Disease response. 
AML: CR, CRi and PR rates as per ELN criteria, 1 month and 6 months after 
MLM-CAR44.1 T-cell infusion. Morphologic leukemia-free state (MLFS) 1, 2 and 
6 months after infusion. 
MM: ORR (sCR, CR, VGPR and PR) as per IMWG criteria, 1, 2 and 6 months 
after T-cell infusion. 
 Overall survival (OS) at 2 years: time from MLM-CAR44.1 T-cell infusion to 
death due to any cause. 
 Disease-free survival (DFR) in AML patients: time from achievement of response 
to relapse or death due to any cause during response. 
 Event Free Survival (EFS) in AML patients: time from the date of MLM-CAR44.1 
T-cell infusion to the date of the earliest of the following: last follow-up, resistance, 
relapse or death due to any cause.  
 Progression-free survival (PFS) in MM patients at 2 years: time from MLM-
CAR44.1 T-cell infusion to progression/relapse or death due to any cause. 
 Duration of response (DOR) in MM patients: time from achievement of response 
to relapse or death from any cause.  


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2. To characterize the in vivo pharmacokinetic profile (engraftment, persistence, trafficking) 
of MLM-CAR44.1 T-cells. 
 Endpoint: Levels of circulating MLM-CAR44.1 T-cells by flow cytometry. 
3. To evaluate the feasibility of treatment with MLM-CAR44.1 T-cells 
The endpoints for assessing feasibility are as follows: 
 Percentage of eligible patients for each indication after screening who undergo 
both lymphocyte apheresis and CAR T-cell infusion. 
 Percentage of patients who are eligible for lymphocyte apheresis and are then 
effectively infused with CAR T-cells. 
4. To evaluate the safety of MLM-CAR44.1 T-cells. 
The endpoints for assessing safety are as follow: 
 Adverse events (any grade) and monitoring for systemic reactions (fever, 
tachycardia, nausea and vomiting, joint pain, skin rash). 
 Absence of RCR monitored by DNA PCR for the Galv gene 3, 6, 12 and 24 months 
after infusion and then yearly as defined in the long-term follow-up. 
 Absence of abnormal MLM-CAR44.1 T-cell clonal expansion monitored by 
clinical and laboratory surveillance, as well as by TCR-Vbeta repertoire study 6 
months after infusion. 
 Suicide gene activation and elimination of transduced cells through administration 
of ganciclovir in case of CRS and other MLM-CAR44.1 T-cell related toxicities. 
5. To conduct an ancillary study including but not limited to the characterization of the 
cellular composition of circulating LNGFR+ (CAR T-cells) and LNGFR- cells, and the 
monitoring of the patient’s immune status through analyses of peripheral blood 
mononuclear cells (PBMC). 
2.2.3 Exploratory objectives 
To evaluate minimal residual disease (MRD): 
 AML: proportion of patients with a molecular CR, 2 months after infusion. 
 MM: proportion of patients with a molecular CR, 3 months after infusion. 
 


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3 
TRIAL DESIGN 
This is a seamless Phase I/IIa, open-label, multicenter clinical trial that combines Phase I dose 
escalation based on toxicity with Phase IIa dose expansion based on antitumor activity. 
 Phase I dose escalation 
A Bayesian Optimal Interval (BOIN) design with cohorts of 3 patients for each indication 
(AML and MM) will be used to single out the MTD based on a target toxicity rate of 30%. 
The BOIN design is a novel Bayesian dose-finding method that minimizes the decision errors 
of dose assignment (for example the chance of exposing patients to overly toxic doses) and thus 
safeguards patient ethics and safety by allowing decisions to be made at any time during the 
study, even when one of the patients in the cohort is not evaluable. This design has performance 
superior to the traditional 3 + 3 design, but shares with it the fact that it can be easily 
implemented, differently from the well-known continual reassessment method (CRM), the gold 
standard design of phase I dose finding. The BOIN design yields an average performance 
comparable with CRM in selecting the MTD but has a lower risk of assigning patients to sub 
therapeutic or overly toxic doses.  
According to this design, the dose assignment for patients who will sequentially enter the study 
is pre-specified and guided by the observed probability of dose-limiting toxicity (i.e. the number 
of patients experiencing DLT at the current dose divided by the total number of patients treated 
at the current dose). The decision boundaries identified by the method, which are optimal in the 
sense that they minimize the chance of incorrect decisions in dose assignments, are 24.7% and 
34.8% (given a tolerance toxicity interval of 20-40%) and the trial will use them as follows: 
 dose escalation, if the observed toxicity rate is smaller than the lower boundary of 
24.7% (E in Table 3-1),  
 dose de-escalation, if the observed toxicity rate is greater than the upper boundary of 
34.8% (D in Table 3-1)),  
 stay at the current dose, if the observed toxicity rate is between 24.7 and 34.8% (S in 
Table 3-1)). 
A Bayesian safety rule will also be applied to eliminate from the trial, and to consider not 
admissible for MTD selection, the doses that are overly toxic (DU in Table 3.1)). This might 
happen when the risk that the toxicity exceeds the target 30% is very high (>95%), given the 
current data (i.e. Pr(toxicity rate>30%|current data)>95%), based on a beta posterior 
distribution. 
The first AML cohort and first MM cohort will be made up of adult patients and will be treated 
with the lowest dose of autologous MLM-CAR44.1 T-cells (0.5 x 10E6/Kg).  
The first three enrolled patients will be treated regardless of indication at lowest dose of 0.5 x 
10E6/Kg one at a time at intervals of at least 36 days to evaluate the occurrence of any toxicity, 
to save the rights, safety, and well-being of the trial patients in this fist-in-human trial setting. 
The second patient will undergo lymphodepleting chemotherapy (from day -5 to day -3 prior 
to CAR T-cell infusion), only once the first patient has completed the 30-day follow-up period 
after CAR T-cell infusion (30 + 5 days). The third patient will undergo lymphodepleting 


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chemotherapy only once the second one has completed the 30-day follow-up after CAR T-cell 
infusion. 
The remaining patients to complete the first cohort for each indication will be treated without 
staggering once the first three enrolled patients have completed follow-up and the dose has 
proven to present a manageable safety profile. 
After all patients of the first AML cohort or first MM cohort (according to BOIN Design) have 
completed the 30-day safety monitoring period, the decision on whether to escalate to the next 
dose will be made together with a Data and Safety Monitoring Board (DSMB) based on the rate 
of dose-limiting toxicities (DLT) and the decision rules presented in Table 3-1. 
The first three patients of the second dose level (1 x 10E6/kg) will be treated with staggering 
regardless of indication only if both indications (AML and MM) have completed the first cohort 
dose, otherwise only the indication that has completed the first cohort dose can be treated. The 
remaining patients to complete the second cohort for each indication will be treated without 
staggering once the first three enrolled patients have completed follow-up and the dose has 
proven to present a manageable safety profile.  
Then, as outlined for the previous cohort level, the first three patients of the third dose level (2 
x 10E6/kg) will be treated with staggering regardless of indication only if both indications 
(AML and MM) have completed the second cohort dose, otherwise only the indication that has 
completed the second cohort dose can be treated. The remaining patients to complete the third 
cohort for each indication will be treated without staggering once the first three enrolled patients 
have completed follow-up and the dose has proven to present a manageable safety profile. 
The second and then third dose will be administered to cohorts of new patients with an 
escalation process that continues until the MTD or the maximum sample size of 15 patients for 
each indication has been reached. The duration of the evaluation period for each patient (30 
days) is based on previous studies with similar cellular products, in which severe CRS (main 
DLT for the study) was observed for up to 7-15 days after infusion. 
0.5 x 10E6/Kg, 1 x 10E6/Kg and 2 x 10E6/Kg are the three doses of autologous MLM-CAR44.1 
T-cells for this study. The recommended Phase IIa dose corresponds to the MTD. If no DLT is 
reported, the dose will be the maximum dose investigated (2 x 10E6/kg). 
 
 


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TABLE 3-1 
TRANSITION RULES FOR EACH DOSE 
 
Number 
of 
patients 
with at 
least 
one 
DLT 
Cumulative Number of Patients 
  
3 
6 
9 
12 
15 
0 
E 
E 
E 
E 
E 
1 
S 
E 
E 
E 
E 
2 
D 
S 
E 
E 
E 
3 
DU 
D 
S 
S 
E 
4 
  
DU 
D 
S 
S 
5 
  
DU 
DU 
D 
S 
6 
  
DU 
DU 
D 
D 
7 
  
  
DU 
DU 
D 
8 
  
  
DU 
DU 
DU 
9 
  
  
DU 
DU 
DU 
10 
  
  
  
DU 
DU 
11 
  
  
  
DU 
DU 
12 
  
  
  
DU 
DU 
13 
  
  
  
  
DU 
14 
  
  
  
  
DU 
15 
  
  
  
  
DU 
E: Escalate to the next higher dose; S: Stay at the same dose; D: De‐escalate to the previous lower dose; 
DU: De‐escalate to the previous lower dose and the current dose will never be used again in the trial (safety rule).  
 Phase IIa dose expansion 
The activity of the MTD will be assessed on 14 patients of each indication (AML and MM 
separately) using Simon’s two-stage design. These cohorts will include the patients enrolled in 
Phase I treated with the MTD, and in the case of AML, also children. 
In the first stage, the hematologic response to treatment of the first 6 patients with each 
indication will be evaluated, and if no response is observed the study will be terminated. 
Otherwise, the study will proceed to stage two and the remaining 8 subjects will be enrolled.  
Transition from Phase I to Phase II will be managed through the submission of a substantial 
amendment to the protocol, which will be evaluated in consultancy with the Data Safety 
Monitoring Board (DSMB). 
 Follow-up 
After being infused with MLM-CAR44.1 T-cells, patients will be monitored closely in the 
hematology or pediatric ward for at least the first 15 days. Follow up will be twice weekly 
through Month 1, weekly for the second month, every two weeks for the third month, monthly 
until Month 6 and then every 3 months until Month 24 to evaluate the safety, antitumor activity 
and feasibility of treatment with MLM-CAR44.1 T-cells. 
 End of study 
The end of study is defined as the last patient’s last visit (LPLV) planned at Month 24 (end of 
primary follow-up), or the time of premature withdrawal. Patients who discontinue primary 
follow-up before month 24 will nevertheless continue to be monitored through a secondary 
follow-up for up to 2 years after MLM-CAR44.1 T-cell infusion to collect data required by 
health authorities (e.g. delayed adverse events). Discontinuation of primary follow-up may be 


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due to progression of disease, treatment failure, relapse after remission, pursuing HSCT while 
in remission, or withdrawal from follow-up.  
Furthermore, evaluations will be performed for up to 15 years (Long-term follow-up) on all 
patients as recommended by health authority guidance for patients treated with gene therapies. 
Separate informed consent/assent forms will be provided for all patients who either complete 
or prematurely discontinue from the study and choose to enroll into the long-term follow-up 
protocol. 
The study can be terminated at any time for any reason by the sponsor or if any of the stopping 
criteria described in Section 5.12 are met. Additional procedures may need to be followed to 
safeguard patient safety.  
The overall study design is shown in Figure 1 


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FIGURE 1 
EURE-CART-1 STUDY DESIGN 
 


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4 
PATIENT SELECTION CRITERIA 
 Target population 
The study population is made up of patients with relapsed/refractory AML or MM expressing 
CD44v6. 
The trial will include pediatric patients with AML (˂ 18 years old) only in Phase IIa, with the 
exception of the center in the Czech Republic, and if all the following criteria have been met 
by adult patients in Phase I: 
1. No SAEs related to lymphocyte apheresis. 
2. No immediate SAEs related to MLM-CAR44.1 T-cell infusion (within 48 hours of 
infusion). 
3. No early SAEs related to infusion (within 30 days). 
4. Evidence of MLM-CAR44.1 T-cell engraftment. 
Should one or more of these criteria not be met, the DSMB (including a pediatrician) will 
evaluate the risk/possible benefit profile of study treatment and provide suggestions on whether 
to include pediatric patients. 
The protocol will be amended to classify pediatric patients according to age groups 
 Inclusion criteria 
Patients must meet all the following inclusion criteria to be eligible for the study. 
1. Written informed consent before any study-related procedure. 
2. Adults and children: 
a) Adults 18 to 75 years old with AML or MM or 18 to 63 years old for CZ site.  
b) Children 1 to 17 years old with AML, only in Phase IIa, except for CZ site.  
3. Confirmed diagnosis of AML or MM as follows: 
a) AML: Primary or secondary AML (any subtype except acute promyelocytic leukemia) 
according to World Health Organization (WHO) classification. 
b) MM with measurable disease defined at least one of the following: 
 Serum M protein ≥ 5g/dL (≥ 5 g/L) 
 Urine M protein ≥ 100 mh/24 hours 
 abnormal serum free light chain (sFLC) assay: FLC ratio <0.26 or >1.65 and/or 
involved Serum free light chain >=100 mg/L 
4. Patients with relapse or refractory disease: 
a) AML patients must be unlikely to benefit from cytotoxic chemotherapy as follows:  
 Leukemia refractory to at least 2 induction attempts (use of a hypomethylating agent 
for at least 4 cycles can be considered a line of treatment). 


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 Leukemia in relapse within 1 year following CR after at least 2 induction attempts 
(use of a hypomethylating agent for at least 4 cycles can be considered a first line of 
treatment). 
 High-risk leukemia in adults according to 2017 ELN (Appendix 9)  in first relapse 
after a hypomethylating agent or a cycle containing cytarabine at a dose ≥ 1g/sqm a 
day (e.g. FLAG-IDA), except for FLT3-mutated AML. 
 High-risk leukemia in children as defined by the Italian Association of Pediatric 
Hematology and Oncology (AIEOP). 
b) Patients with MM must have a relapse or refractory disease after at least 4 different 
prior treatments in 3 treatment lines, or 4 treatments in 2 treatment lines in case of early 
relapsing patients (relapse in less than 1.5 years). Treatments include: 
 Proteasome inhibitor 
 High-dose alkylating agent if patients less than 70 years old 
 Immunomodulatory drug (IMID) 
 A monoclonal antibody (i.e. anti CD38 monoclonal antibody) 
5. Positive CD44v6 expression on tumor cells by flow cytometry ≥ 20%. 
6. Eastern Cooperative Oncology Group (ECOG) performance status 0-2. 
7. Life expectancy of at least 12 weeks. 
8. Adequate organ function: 
a) Alanine aminotransferase (ALT) level within 2.5 times the institutional upper limit of 
normal (ULN). 
b) aspartate aminotransferase (AST) level ≤ 2.5 x ULN. 
c) Total bilirubin level ≤ 1.5 x ULN, or ≤ 2.5 x ULN in case of history of Gilbert’s disease. 
d) Serum creatinine ≤ 2.0 mg/dL or a calculated or measured creatinine clearance ≥ 
45mL/min. 
e) Corrected Diffusing Capacity of Carbon Monoxide (DLCO) (via Dinakara Equation) or 
forced expiratory volume in one second (FEV1) of  66% without dyspnea on slight 
activity after hemoglobin correction. 
f) Left ventricular ejection fraction ˃ 45%. 
9. Recovery from toxicities of clinical consequence attributed to previous chemotherapy to 
CTCAE v5.0 Grade 1 (i.e., certain toxicities such as alopecia will not be considered in this 
category). 
10. Ability to comply with study procedures, including hospitalization and protocol-specified 
acquisition of blood and/or bone marrow specimens. 
11. Willing to be followed up long term, i.e. a 15-year follow up as required by health 
authorities for cell and gene therapy products. 
12. Women of childbearing potential must test negative for pregnancy at enrolment and during 
the study and agree to use an effective method of contraception until 1 year after CAR T-


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cell infusion. Male patients with partners of childbearing potential must be either 
vasectomized or agree to use a condom in addition to having their partners use another 
method of contraception resulting in a highly effective method of birth control. See Section 
6. 7 for details. 
 
 Exclusion criteria 
4.3.1 At screening 
Patients must meet none of the following exclusion criteria to be eligible for the study: 
1. History of or candidate for allogeneic stem cell transplantation. 
2. Cardiovascular, pulmonary, renal, and hepatic  functions that in the judgment of the 
investigator are insufficient for the patient to undergo investigational CAR T-cell therapy. 
3. Any history of or suspected current autoimmune disorders (apart from vitiligo, resolved 
childhood atopic dermatitis, Graves’ disease clinically controlled). 
4. History of rheumatologic disorders requiring specific treatment at any time in the patient’s 
medical history. 
5. Second primary malignancy that requires active therapy. Adjuvant hormonal therapy is 
allowed.  
6. Known or suspected central nervous system (CNS) leukemia. 
7. Presence or history of myeloid sarcoma or any extramedullary mass. 
8. Any medical or psychiatric condition that may limit compliance or increase safety risks, 
such as:  
a) Active uncontrolled infection (including, but not limited to viral, bacterial, fungal, or 
mycobacterial infection). 
b) Patients with known multiple antibiotic resistant infections in their clinical history. 
c) Known human immunodeficiency virus infection, active or chronic hepatitis B or C 
infection. 
d) Grade 3 or 4 bleeding. 
e) Uncontrolled hypertension (systolic pressure > 180 mm Hg or diastolic pressure > 100 
mm Hg). 
f) Clinically significant arrhythmia, clinically significant baseline QTcF, or QTcF > 480 
msec. 
g) Unstable angina. 
h) Myocardial infarction within 6 months prior to study entry. 
i) Clinically significant heart disease (e.g. CHF NYHA III or IV, unstable coronary artery 
disease, myocardial infarction < 6 months prior to study entry). 
j) Pregnancy or breastfeeding. 


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k) Major surgery or trauma within 4 weeks before enrollment. 
l) Dementia or altered mental status that would preclude sufficient understanding to 
provide informed consent. 
Once all other eligibility criteria are confirmed, a lymphocyte apheresis product of non-
mobilized cells must be received and accepted by the manufacturing site. Note: the lymphocyte 
apheresis product will not be shipped or assessed for acceptance by the manufacturing site until 
documented confirmation of all other eligibility criteria has been received 
 Prior to lymphocyte apheresis (week -9 to -7). 
The following criteria must be met: 
1. Peripheral blast count ≤ 20,000/mm3 (AML). 
2. No treatment with any other investigational agent in the previous 4 weeks. 
3. No treatment with an immunostimulatory agent (IMIDs are allowed) or any cell therapy in 
the previous 30 days. 
4. Negative to the following tests: HCV (Antibody, NAT), HIV 1-2 (p24, AB, Ag and NAT), 
total Ig Treponema Pallidum (if positive perform specific test), Australia HBsAg, total anti 
HB core Ab (if positive perform HBV DNA NAT), mycoplasma (PCR or IgM) and HTLV 
I-II. 
5. No treatment with steroids at least 3 days before lymphocyte apheresis 
 Prior to lymphodepleting chemotherapy/ MLM-CAR44.1 T-cell 
infusion (day -5 to day -3)  
The following criteria must be met: 
1. Evidence of active disease at the beginning of lympho-depleting chemotherapy  
2. The following medications are excluded and should not be administered: 
a. Monoclonal antibodies in the 8 weeks prior to MLM-CAR44.1 T-cell infusion are 
prohibited. 
b. Salvage chemotherapy is allowed (e.g. clofarabine, cytosine arabinoside > 100 mg/m2, 
anthracyclines, cyclophosphamide, proteasome inhibitors, IMIDs) but must be stopped 
> 2 weeks prior to lymphodepleting chemotherapy 
c. Granulocyte colony stimulating or granulocyte-macrophage colony stimulating factor 
in the 2 weeks prior to study CAR T-cell administration. 
d. Immunosuppressant medications in the 2 weeks prior to CAR T-cell administration. 
e. Cytosine arabinoside < 100 mg/m2/day must be stopped > 1 week prior to MLM-
CAR44.1 T-cell infusion. 
f. Therapeutic systemic doses of steroids must be stopped > 72 hours prior to product 
infusion. However, < 12 mg/m2/day hydrocortisone or equivalent are allowed as 
physiological replacement doses of steroids. 
g. Hydroxyurea must be stopped > 72 hours prior to MLM-CAR44.1 T-cell infusion. 


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3. No cardiovascular, pulmonary, renal, and hepatic  functions that in the judgment of the 
investigator are insufficient for the patient to undergo investigational CAR T-cell therapy. 
4. Female patients of childbearing potential must have a negative pregnancy test within 24 
hours prior to starting lymphodepleting therapy. 


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5 
EXPERIMENTAL PRODUCT 
 Study drug information 
The medicinal product consists of autologous T-cells (MLM-CAR44.1 T-cells) genetically 
modified by retroviral vector LTK-S44v6ΔNL to express the CD44v6 CAR and HSV-TK Mut2 
genes. The drug product is defined as the frozen MLM-CAR44.1 T-cell suspension formulated 
in frozen medium at the concentration of 1.0-10 x 10E6 cells/mL and ready for intended medical 
use. The product is patient specific as it is prepared starting from lymphocytes of the patient 
collected through lymphocyte apheresis. See Section 1.2 for details about drug description. 
 Study drug supply 
The cryopreserved MLM-CAR44.1 T-cell product will be shipped to the trial center from the 
manufacturing site. Upon receipt, the T-cell product must be received by designated personnel 
at the study site, handled and stored safely and properly, and kept in a secured location to which 
only the investigator and designated site personnel have access. Any damaged or unusable cell 
product must be documented and notified to the study Sponsor. 
 Study drug accountability, disposal and destruction 
Traceability between the patient’s autologous apheresis and the MLM-CAR44.1 T-cell product 
will be compliant with Regulation (EC) 1394/2007, Directive 2004/23/EC (including all related 
directives) as well as EU “Detailed guidelines on good clinical practice specific to advanced 
therapy medicinal products.” The data contributing to the full traceability of the cells are stored 
for a minimum of 30 years. A unique patient identifier will maintain a chain of identity between 
the apheresis product and the MLM-CAR44.1 T-cell batch, and the link between patient identity 
and unique patient identifier will be confirmed prior to infusion. 
The investigator or designee must maintain an accurate record of the shipment and dispensing 
of the MLM-CAR44.1 T-cell product in a drug accountability log. Drug accountability will be 
reviewed by the monitor during site visits and at the completion of the study. 
The investigator will dispose of used and unused MLM-CAR44.1 T-cell product, packaging, 
product labels as per local institutional standard operating procedures, and return a copy of the 
completed drug accountability log to the study monitor. 
See the MLM-CAR44.1 Study Manual for details. 
 Formulation, packaging, labelling and storage 
Each infusion bag will contain 10-50 mL of cells containing a cell dose of 0.5 or 1 or 2 x 
10E6/Kg of autologous CD44v6 CAR T-cells. Each infusion bag will be labelled according to 
GMP for advanced therapy and GCP guidelines. Labels will include at least the following 
information: protocol name, the Principal Investigator’s name, batch number, patient code, 
storage conditions, manufacturing date, total number of cells, total volume, concentration, route 
of administration, Sponsor’s name, “For clinical trial use only” and the expiry date. Text might 
be in different order on the labels and will be adapted to the country specific local requirements. 
Additional label elements required by local regulations will also be included. Prior to the 


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infusion, two individuals will confirm identity according to local institutional guidelines to 
ensure that the patient receives only their autologous product. 
The investigational product must be stored safely and properly. The cryopreserved cell product 
should be kept in the vapor phase of liquid nitrogen until infusion. The product should be 
thawed near the patient’s bedside so the time between thawing and completion of the infusion 
does not exceed 60 minutes, thus maintaining maximum product viability. After thawing, the 
MLM-CAR44.1 T-cell product should NOT be washed prior to infusion. If the product bag 
appears to be damaged or is leaking, the product should not be infused and should be disposed 
of according to local institutional procedures. 
See the MLM-CAR44.1 Study Manual for details. 
 Dosage schedule  
Patients will receive a single dose of MLM-CAR44.1 T-cells: 0.5 x 10E6/Kg or 1 x 10E6/Kg or 
2 x 10E6/Kg according to the BOIN design in Phase I, and a dose corresponding to the MTD in 
Phase IIa. 
5.5.1 Dose modification 
Dose escalation or de-escalation will take place during Phase I following the BOIN design 
outlined in Section 3 and considering the following dose limiting toxicities.  
 Grade 4 CRS 
 Grade 3 or higher CRS not responsive to therapy with steroids and/or 
tocilizumab/siltuximab within 24 hours  
 NCI-CTC grade 3 or higher toxicity at least possibly related to treatment with MLM-
CAR44.1 T-cells excluding hematological toxicities 
 Skin toxicities:  
o 
NCI-CTC grade 3 or higher erythroderma (generalized exfoliative dermatitis) 
o 
Stevens-Johnson syndrome 
o 
Toxic Epidermal Necrolysis (TEN) 
o 
Any other grade 3 or higher skin toxicity histologically confirmed as being related 
to CAR T-cell treatment 
 Grade 3 or higher neurotoxicity 
Dose modifications are not applicable in Phase IIa.  
5.5.2 Treatment duration 
The MLM-CAR44.1 T-cell product will be administered as a single intravenous infusion. 
 Lymphocyte apheresis 
The first step in manufacturing CAR T-cells is to collect autologous lymphocytes by apheresis. 
Patients will undergo non-stimulated lymphocyte apheresis at least 7 weeks prior to MLM-
CAR44.1 T-cell infusion to allow cell production, quality and release controls, and completion 
of lymphodepleting chemotherapy. All efforts will be made to perform CAR T-cell 
manufacturing within 5-6 weeks after lymphocyte apheresis. 


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Apheresis 
should 
be 
scheduled 
prior 
to 
the 
administration 
of 
chemotherapy, 
immunomodulators, or steroids to ensure an adequate absolute lymphocyte count (ALC). Prior 
treatment with an immunostimulatory agent (IMIDs are allowed) or any cell therapy in the 30 
days prior to lymphocyte apheresis is prohibited. Patients may be required to undergo multiple 
apheresis procedures to collect a target of 7 × 10E8 CD3+ cells or a minimum of 5 x 10E8, or, 
when it is not possible, approximately 10 x 10E9 WBC, for the preparation of the investigational 
product. AML patients should however present a peripheral blast count ≤ 20,000/mm3 at time 
of lymphocyte apheresis. 
The lymphocyte apheresis product will not be shipped or assessed for acceptance by the 
manufacturing site until documented confirmation of eligibility criteria has been received. See 
section 4.4. Full details of apheresis requirements, as well as of the procedures for procurement, 
handling and shipment of the apheresis product are present in the MLM-CAR44.1 Study Manual. 
In most patients undergoing CAR T-cell therapy, lymphocyte apheresis is well tolerated, and 
adequate numbers of lymphocytes are collected (Allen et al., 2017). Some patients may however 
experience adverse effects including fatigue, nausea, dizziness, feeling cold, and tingling in the 
fingers and around the mouth. More serious complications, including abnormal heart rate and 
seizures, can occur but are extremely rare. A retrospective analysis of 15,763 apheresis 
procedures found only 59 moderate-to-severe adverse events (AEs) (0.37%), including 
dizziness or fainting episodes (0.12%), citrate toxicity (0.02%), a combination of 
dizziness/fainting and citrate toxicity (0.11%), vascular injuries (0.07%), and miscellaneous 
events (0.04%) (Yuan et al., 2010). 
 Salvage chemotherapy  
Patient can undergo a salvage chemotherapy (e.g. clofarabine, cytosine arabinoside > 100 
mg/m2, anthracyclines, cyclophosphamide, proteasome inhibitors, IMIDs) after lymphocyte 
apheresis and prior to lymphodepleting chemotherapy.  
Salvage chemotherapy must be stopped > 2 weeks prior to lymphodepleting chemotherapy, so 
it should last at most 4 weeks. 
It is recommended to communicate to Sponsor the salvage chemotherapy intended to be 
administered. 
 Lymphodepleting chemotherapy 
Lymphodepleting chemotherapy prior to T-cell infusion has been shown to favor T-cell 
engraftment and proliferation. This preconditioning creates space for the expansion of infused 
cells, limits the competition for homeostatic gamma chain cytokines IL-7 and IL-15, depletes 
regulatory T cells, and activates the innate immune system.  
Patients must meet the eligibility criteria for lymphodepleting chemotherapy stated in Section 
4.5. Should this not occur, the Investigator is to delay lymphodepleting chemotherapy for up to 
60 days until the patient’s conditions are deemed suitable for treatment. Any delay of more than 
1 week must be discussed with the Sponsor in order to evaluate how to proceed with the 
treatment of the next patient, who has to be infused at interval of 36 days from the previous 
one. 


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The Investigator must inform the Sponsor of the start date of lymphodepleting chemotherapy 
to confirm the delivery date of the MLM-CAR44.1 T-cells.  
The following criteria must be met: 
a. Evidence of active disease at the beginning of lympho-depleting chemotherapy. 
b. Salvage chemotherapy (e.g. clofarabine, cytosine arabinoside > 100 mg/m2, 
anthracyclines, cyclophosphamide, proteasome inhibitors, IMIDs) must have been 
stopped > 2 weeks prior to lymphodepleting chemotherapy. 
c. Female patients of childbearing potential must have a negative pregnancy test within 
24 hours prior to starting lymphodepleting therapy. 
d. From October through May, patients should undergo a rapid influenza diagnostic test 
within 10 days prior to lymphodepleting chemotherapy. If the patient is positive, 
oseltamivir phosphate or zanamivir should be administered for 10 days as preventative 
treatment and the test repeated prior to lymphodepleting chemotherapy. If flu-like 
symptoms are present, lymphodepleting chemotherapy should be delayed until the 
patient is asymptomatic. 
Patients will undergo planned lymphodepleting chemotherapy with: cyclophosphamide i.v. 
(500 mg/m2) and fludarabine i.v. (30 mg/m2) daily from day -5 to day -3. 
NOTE: Patients should be closely monitored for tumor lysis syndrome before and after 
lymphodepleting chemotherapy through blood tests for potassium and uric acid (see section 
11.6.1.5). Patients with elevated uric acid or high tumor burden should receive prophylactic 
allopurinol, or a non-allopurinol alternative 
 Treatment with MLM-CAR44.1 T-cells 
The MLM-CAR44.1 T-cell product will be released by the manufacturing facility and shipped 
to the study site approximately 4 weeks after the start of manufacturing. Details of the storage 
and handling requirements for the cryopreserved MLM-CAR44.1 T-cell product are available 
in the MLM-CAR44.1 Study Manual. 
The following should be considered prior to MLM-CAR44.1 T-cell infusion: 
 
1. Patient should not present a significant worsening of clinical status or laboratory 
abnormalities that would, in the opinion of the Investigator, increase the risk of adverse 
events associated with T-cell treatment or compromise the likelihood of completing the 
study protocol. MLM-CAR44.1 T-cell infusion should be delayed until clinically 
appropriate for up to 10 days. 
Patients with significant progression of disease following lymphodepleting chemotherapy 
are at high risk of severe CRS and must not receive T-cell infusion. 
2. T-cell infusion should be delayed for up to 10 days in case of significant unresolved 
toxicities from preceding lymphodepleting chemotherapy, especially toxicities requiring 
supplemental oxygen, uncontrolled cardiac arrhythmia, hypotension requiring vasopressor 
support and uncontrolled active infection. 
 


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3. The following medications must be stopped prior to MLM-CAR44 T-cell infusion at the 
following times: 
a. Monoclonal antibodies: 8 weeks before.  
b. Granulocyte colony stimulating or granulocyte-macrophage colony stimulating 
factor and immunosuppressant medications: 2 weeks before. 
c. Cytosine arabinoside < 100 mg/m2/day: 1 week before. 
d. Immunosuppressant medications in the 2 weeks prior to CAR T-cell 
administration 
e. Therapeutic systemic doses of steroids must be stopped > 72 hours prior to 
product infusion. However, < 12 mg/m2/day hydrocortisone or equivalent are 
allowed as physiological replacement doses 
f. Hydroxyurea should be stopped > 72 hours prior to MLM-CAR44.1 T-cell 
infusion 
g. Ganciclovir/valganciclovir should be stopped at least 48 hours prior to MLM-
CAR44.1 T-cell infusion  
5.9.1 Premedication 
Systemic corticosteroids other than physiologic replacement of hydrocortisone should not be 
administered except in the case of life-threatening emergency as this may negatively affect 
MLM-CAR44.1 T-cell expansion and function. Patients should be pre-medicated with 
paracetamol and an H1 antihistamine for infusion side effects such as fever, chills and nausea. 
5.9.2 MLM-CAR44.1 T-cell infusion 
Detailed instructions for the preparation and administration of the MLM-CAR44.1 T-cell 
product are available in the MLM-CAR44.1 Study Manual.  
The CAR T-cell product should not be washed after cell thawing, and the infusion should be 
completed within 60 minutes of thawing to maintain cell viability.  
Patients will receive one dose of MLM-CAR44.1 T-cells during a single intravenous infusion, 
preferably through a central line and latex free i.v. tubing without a leukocyte filter 
(approximately 10 to 20 mL per minute). The tubing setup should be fitted with a connector 
and an attached supplemental saline bag to be used after the initial infusion is completed 
allowing any remaining product remaining in the bag and tubing to be recovered and infused. 
Vital signs (temperature, heart rate, diastolic and systolic blood pressure) are to be taken: 
 prior to MLM-CAR44.1 T-cells infusion 
 during infusion 
 at the end of infusion 
 after the infusion, approximately every 15 minutes for one hour and then every hour 
for the next two hours, or until these signs are satisfactory and stable.  
Emergency medical equipment should be available during the infusion in case the patient has a 
significant infusion reaction such as CRS or anaphylaxis. Tocilizumab/siltuximab must be 
available on site prior to CAR T-cell infusion to manage CRS and supportive therapy such 


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as oxygen, bronchodilators, epinephrine, antihistamines, and corticosteroids. These 
medications should be given according to the safety monitoring guidelines in Section 11.6.1. 
 Rescue medication 
Rescue medications are those given for toxicity due to MLM-CAR44.1 T-cell therapy. 
Tocilizumab (recommended dose 8 mg/kg i.v.), siltuximab (11 mg/kg IV over 1 hour) and 
steroids may be used for the treatment of suspected severe CRS as described in Section 11.6.1. 
Prior to MLM-CAR44.1 T-cell infusion, the site must confirm that two doses of tocilizumab 
are on site and available for administration.  
Significant off-tumor toxicity can be controlled by setting off suicide gene activation through 
administration of ganciclovir 10 mg/kg/day divided into 2 administrations, or valganciclovir 
900 mg twice per day orally for 14 days. Ganciclovir should be avoided right before and right 
after the infusion: a wash out of 48 hours is recommended. There is no issue in using others 
drugs such as Foscarnet if needed in a timeframe closer to the infusion period. Ganciclovir is 
commonly used in the field of congenital and acquired pathologies causing immunodepression 
for the prevention and therapy of cytomegalovirus reactivation and for the treatment of GvHD 
in patients infused with lymphocytes genetically modified with HSV-tk. The dose in both cases 
is equal to 10 mg/kg/day, with reduced dosage in case of impaired renal function. Other rescue 
medications such as the ones indicated previously can continue to be administered until the 
effects of suicide gene activation are clinically relevant. The use of ganciclovir can be 
accompanied by side effects such as marrow depression, gastrointestinal toxicity and impaired 
renal function. However, these side effects occur especially after long-term administration. 
Based on the clinical judgment of the Investigator, and in the presence of the following 
toxicities at least possibly related to MLM CAR44.1 T-cells, ganciclovir may be administered 
to set off suicide gene activation: 
 Grade 3 or higher neurotoxicity 
 Grade 4 CRS 
 Grade 3 CRS not responsive to steroids after tocilizumab failure within 24 hours (see 
details in Table 11-3). 
 Grade 3 erythroderma not responsive to steroids (methylprednisolone 2mg/kg for 72 
hours or rapidly evolving) 
 Grade 2 or higher bullous dermatitis 
 Any grade Stevens Johnson Syndrome 
 Any evidence for Toxic Epidermal Necrolysis (TEN) 
 Any other grade 3 or higher toxicity suspected of being related to CAR T-cell 
treatment not responsive to appropriate first-line treatment or judged to be potentially 
life threatening by the investigator. Patients who remain monocytopenic after MLM-
CAR44.1 T-cell therapy should be considered immune-depressed. Suicide gene 
activation should thus also be considered in case of grade 3-4 potentially life-
threatening infections. 
In case of administration of ganciclovir/valganciclovir for activation of suicide gene, an 
immune phenotype analysis of MLM-CAR44.1 T-cells by flow cytometry (peripheral blood) 
must be performed at the following time-points: 


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 before ganciclovir/valganciclovir administration 
 4 days after ganciclovir/valganciclovir administration 
 one day after the discontinuation of ganciclovir/valganciclovir administration 
It is strongly recommended to share with Sponsor the decision to activate suicide gene. 
All rescue medications must be listed on the concomitant medication CRF. Suicide gene 
activation shall be reported immediately to the Sponsor and DSMB. 
 Concomitant therapy 
Clinically significant prescription and nonprescription medications will be recorded in the 
patient’s medical record and in the CRF at every visit following the screening visit. 
Concomitant therapies to be avoided during or shortly prior to MLM-CAR44.1 T-cell therapy 
are outlined in Section 5.9. 
 Stopping rules 
Early discontinuation of the study will be based on review of SAEs. Only unexpected SAEs 
related to treatment with MLM-CAR44.1 T-cells should be considered. The review of these 
adverse events, and any decision to prematurely stop patient enrollment, will be discussed with 
the DMSB and reviewed by the IRB/IEC.  
Premature termination may occur because of a regulatory authority decision, change in opinion 
of the IRB/IEC or DMSB, or may be related to issues concerning cell product generation or 
safety at the discretion of the study investigators. 
The study will be terminated prematurely also if no disease response to treatment is detected in 
the first 6 patients with each indication evaluated in Phase IIa (first stage of Simon’s two-stage 
design). 
5.12.1 Criteria for interrupting or discontinuing the study 
5.12.1.1 Phase I 
 Grade 4 or higher toxicity that is unmanageable, unexpected and unrelated to 
chemotherapy and attributable to MLM-CAR44.1 T-cell therapy will be promptly 
assessed with the DSMB (see section 10). Decision to interrupt or discontinue the 
study will be taken case by case, based on risk/benefit assessment. CRS, 
neurotoxicity, ICU admission, dialysis and mechanical ventilation may be required 
and are not considered unexpected.  
5.12.1.2 Phase IIa 
 Uncontrolled T-cell proliferation in any patient beyond 8 weeks from CAR T-cell 
infusion that does not respond to management. 
 Detectable replication competent retrovirus (RCR) during the study in any patient. 


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 The Investigator, Sponsor, DMSB, or any independent review board or regulatory 
body decides for any reason that patient safety may be compromised by continuing 
the study. 
 Lack of disease response. 
The study will be interrupted pending investigation and possible protocol amendment if any 
patient experiences any of the following events within three weeks of CAR T-cell infusion: 
 Grade 4 toxicity that is unmanageable, unexpected and unrelated to chemotherapy 
and attributable to MLM-CAR44.1 T-cell therapy. CRS, neurotoxicity, ICU 
admission, dialysis and mechanical ventilation are not considered unexpected. The 
same may apply to grade 4 liver toxicity, nephrotoxicity and other organ 
involvement. 
Death suspected of being related to MLM-CAR44.1 T-cell therapy. 
 
 


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6 
STUDY ASSESSMENTS AND PROCEDURES 
The Tables provided in Section 7 present all the planned assessments and procedures. Patients 
who are prematurely discontinued from the study will continue to be monitored through a 
secondary follow-up to collect data required by health authorities (e.g. delayed adverse events, 
etc.). 
 Acute Myeloid Leukemia 
Table 7-1 presents all the planned assessments and procedures for patients with AML. 
 
6.1.1. Screening (Week -10 to Week -8) and enrollmentIn Phase I, the first three enrolled 
patients (regardless of indication) for each dose will be treated one at a time at intervals of at 
least 36 days. Each of these three patients will undergo lymphodepleting chemotherapy only 
once the previous patient has completed the 30-day follow-up or until the previous patient 
experiences a DLT within the completion of 30-day follow-up period (see section 9.1 for the 
definition of “evaluable” patient). The remaining patients for each dose will be enrolled once 
the dose has proven to be safe in the first three enrolled patients. 
Children with AML will be enrolled in Phase IIa only, except for CZ site. 
Any screening assessment that is done outside the screening window must be repeated prior to 
apheresis.Each patient will be identified by a unique patient number, assigned centrally through 
a web interface. This patient number will be used for the entire study. See the MLM-CAR44.1 
Study Manual for details on enrollment procedures. 
The screening and leukemia workup include the following assessments: 
 Evaluation of CD44v6 expression by tumor cells (see section below) 
 Demography (year of birth, age, sex, childbearing status of females, race) 
 Inclusion/exclusion criteria 
 Medical history, AML diagnosis/history 
 Prior/concomitant medications and antineoplastic therapies 
 Bone marrow aspirate/biopsy and peripheral blood draw for morphology, 
immunophenotyping, cytogenetics, molecular cytogenetics by means of fluorescence 
in situ hybridization (FISH) and MRD 
 Brain MRI scan 
 Extramedullary disease (physical exam and CNS system assessment) 
 Complete physical examination including height and weight 
 Vital signs (temperature, heart rate, diastolic and systolic blood pressure) 
 ECOG performance status  
 DLCO or FEV1 
 Pulse oximetry 
 MUGA or ECHO 
 ECG 
 Serum pregnancy test for women of childbearing potential 


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 Local laboratory evaluations (hematology, clinical chemistry, urine analysis, 
creatinine clearance) 
 CD3/mcl count in peripheral blood 
 Coagulation panel  
 Serum β2-microglobulin and LDH 
 Viral serology (HAV, HBV, HCV, HIV, VZV, HSV 1-2, HSV-6, CMV, EBV) 
mycoplasma and toxoplasma. Not required if performed as part of clinical routine 
within 4 weeks prior to informed consent. 
 Adverse events 
 Absence of active infections and/or viral infections requiring the use of ganciclovir 
 
6.1.1.1 Re-screening 
In case of screening failure, for example for an adverse event, that has delayed the execution of 
screening assessments, the investigator can re-include the patient in the clinical trial only if the 
following conditions are satisfied: 
1. Positivity to CD44v6 marker at first screening 
2. Absence of active infections and/or viral infections requiring the use of ganciclovir 
 
Re-screened patient will be identified with another screening code, but he/she will be linked 
with the previous screening code. The outcome of the re-screening phase will be considered for 
the purposes of the study. 
6.1.1.2 Evaluation of CD44v6 expression on tumor cells  
In study Phase I, CD44v6 expression on tumor cells in peripheral blood and bone marrow will 
be evaluated by a centralized laboratory at the San Raphael Hospital. The same assessment will 
also be performed at each local laboratory to set-up a validation process for CD44v6 marking. 
AML patient’s enrolment: CD44v6 positivity is required for bone marrow and peripheral blood 
sample, a positivity at least for the peripheral blood sample is required for some AML with 
extra-medullar involvement such as cutaneous localization (see M4 AML).  
The threshold value of CD44v6 positivity on tumor cells should be ≥ 20%. 
In case of the result of central laboratory is different from that of local laboratory, the central 
assessment will be considered. Details on sample collection are provided in the MLM-CAR44.1 
Study Manual. 
6.1.2 Lymphocyte apheresis (Week -9 to Week -7)  
Lymphocyte apheresis is described in Section 5.6, and full details are provided in the MLM-
CAR44.1 Study Manual. The procedure should take place no longer than 7 weeks prior to MLM-
CAR44.1 T-cell infusion and prior to the administration of chemotherapy, immunomodulators, 
or steroids to ensure an adequate ALC.  
Patients may be required to undergo multiple apheresis procedures to collect a target of 7 × 
10E8 CD3+ cells or a minimum of 5 x 10E8, or when is not possible approximately 10 x 10E9 
WBC, for the preparation of the investigational product. Patients should however present a 
peripheral blast count ≤ 20,000/mm3 at time of lymphocyte apheresis. 


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Prior to lymphocyte apheresis, patients should undergo a check of inclusion/exclusion criteria 
and concomitant therapies. Only after informed consent and confirmation of all eligibility 
criteria will the patient’s apheresis product be shipped to the manufacturing facility. 
6.1.3 Pre-chemotherapy evaluation visit (Week -2) 
Before lymphodepleting chemotherapy begins, the patient will undergo blood collection for 
safety assessments and other assessments as listed below: 
 Check of inclusion/exclusion criteria 
 Physical examination and vital signs 
 Concomitant medications 
 Hematology, clinical chemistry 
 Disease evaluation in bone marrow and peripheral blood (morphology and flow 
cytometry) 
 Adverse events 
 Rapid influenza test (within 10 days of infusion) If the patient is positive, oseltamivir 
phosphate or zanamivir should be administered for 10 days as preventative treatment 
prior to CAR T-cell infusion 
At the discretion of the Investigator, assessments may be repeated for up to 60 days until the 
patient is considered suitable for lymphocyte depleting chemotherapy. 
6.1.4 Lymphodepleting chemotherapy (Day -5 to Day -3) 
Patients will undergo lymphodepleting chemotherapy with cyclophosphamide i.v. (500 mg/m2) 
and fludarabine i.v. (30 mg/m2) daily from day -5 to day -3. 
The following assessments should be performed within 24 hours prior to starting 
lymphodepleting chemotherapy. 
 Physical examination and vital signs 
 Weight 
 Urine pregnancy test for women of childbearing potential 
 Concomitant medications 
 Adverse events 
 Verification of criteria detailed in Section 5.8 
6.1.5 Pre-infusion visit (Day -1) 
The following exams must be performed one day prior to MLM-CAR44.1 T-cell infusion. 
 Physical exam, weight and vital signs 
 ECOG performance status assessment 
 Hematology, clinical chemistry and coagulation panel 
 MLM-CAR44.1 T-cell infusion prerequisites (see Section 5.9) 
 Concomitant medications 
 Adverse events 


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6.1.6 MLM-CAR44.1 T-cell infusion (Day 0) 
See Section 5.9 for details of MLM-CAR44.1 T-cell infusion. Patients will undergo the 
following assessments the day of infusion (prior to infusion): 
 Physical examination, weight, vital signs pre-, during and post-infusion as indicated 
in Section 5.9.2 
 Pulse oximetry 
 ECOG performance status 
 Hematology and clinical chemistry 
 ECG 
 Concomitant medications 
 MLM-CAR44.1 PK (pre-infusion) by flow cytometry in peripheral blood 
 MLM-CAR44.PK (pre-infusion) by PCR in peripheral blood 
 Blood sample collection (pre-infusion) for ancillary studies 
 Blood sample collection for RCR monitoring (pre-infusion) by DNA PCR 
 Plasma sampling (pre-infusion) for cytokine monitoring  
 Adverse events 
 See Section 11.6.1 for safety monitoring guidelines following infusion 
6.1.7 Post-infusion visits: D4±1d, D7±1d, D11±1d, D14±1d, D18±1d, D21±1d, 
D±25±1d and D28±1d (Month 1) 
Patient hospitalization with close monitoring is recommended for at least 15 days after CAR-
T-cell infusion. See Section 11.6.1 for details about safety monitoring. 
The following assessments will be performed at the post-infusion visits: 
 Physical examination and vital signs: all visits 
 Pulse oximetry: D4, D7, D11, D14 
 ECOG performance status: D7, D14, D21, D28 
 Hematology and clinical chemistry: all visits 
 Coagulation panel: D7, D14, D28 
 Urine analysis: D7 and D21 
 Concomitant medications: all visits 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry: D7, D14, D21, D28 
 MLM-CAR44.1 PK (peripheral blood) by PCR: D7, D14, D21, D28 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry: Day 28 
 Blood sample collection for ancillary studies: D7, D14, D21, D28 
 Plasma sampling for cytokine monitoring: D7, D14, D21, D28  
 Adverse events: all visits 
 Weight: D28 
 See Section 11.6.1 for safety monitoring guidelines following infusion 
Assessments will include an evaluation of disease response as follows: 
 Tumor cell assessment by flow cytometry in peripheral blood on D7, D14, D21 and 
D28 


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 Peripheral blood for morphologic blasts on D28 
 Bone marrow aspirate/biopsy – morphology and flow cytometry on D28 
 MRD in bone marrow aspirate if CR/CRi is confirmed 
 Extramedullary disease on D28 
 Response assessment by Investigator: D28 
Frequent monitoring of serum CRP, ferritin and cytokines should be considered during CRS of 
any severity, especially in case of persisting fever, hemodynamic instability, respiratory distress 
and rapid clinical deterioration. See Section 11.6.1 for CRS management. 
6.1.8 Post-infusion visits: Weekly from W5±3d to W8±3d (Month 2) 
From Week 5 to Week 8 patients will undergo one or more of the following assessments during 
weekly visits: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 60 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 60 only) 
 Sample collection (peripheral blood) for ancillary studies (Day 60only) 
 Plasma sampling for cytokine monitoring (Day 60 only) 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry (Day 60 only) 
 Concomitant medications 
 Adverse events 
 Disease assessments at Week 8 only: 
o 
Tumor cell assessment by flow cytometry in peripheral blood 
o 
Peripheral blood for morphologic blasts 
o 
Bone marrow aspirate/biopsy for morphology and flow cytometry 
o 
MRD in bone marrow if CR or CRi is confirmed 
o 
Extramedullary disease 
o 
Disease response by Investigator 
6.1.9 Post-infusion visit: W10±3d and W12±3d (Month 3) 
The following assessments will be performed on Week 10 and Week 12: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 Urine analysis at Week 10 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 90 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 90 only) 
 Blood sample collection for ancillary studies (Day 90 only) 
 Plasma sampling for cytokine monitoring (Day 90 only) 
 RCR monitored by DNA PCR (Day 90) 
 Concomitant medications 


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 Adverse events 
 Disease assessments at Week 12 only: 
o 
Tumor cell assessment by flow cytometry in peripheral blood 
o 
Peripheral blood for morphologic blasts 
o 
Extramedullary disease 
o 
Disease response by Investigator 
6.1.10 Post-infusion visits: Monthly from M4±14d to M6±14d 
The following exams will be performed monthly: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 180 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 180 only) 
 Blood sample collection for ancillary studies (Day 180 only) 
 Plasma sampling for cytokine monitoring (Day 180 only) 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry (Day 180 only) 
 RCR monitored by DNA PCR (Day 180 only) 
 Concomitant medications 
 Adverse events 
One or more of the following disease assessments will be performed monthly: 
 Tumor cell assessment by flow cytometry in peripheral blood at Month 6 
 Peripheral blood for morphologic blast (M4-M6) 
 Bone marrow aspirate/biopsy at Month 6 for morphology and flow cytometry 
 MRD in bone marrow at Month 6 if CR or CRi is confirmed 
 Extramedullary disease (M4-M6) 
 Response assessment by Investigator 
6.1.11 Post-infusion visits: M9±14d, M12±14d, M15±14d, M18±14d, M21±14d, M24 
±14d (end of primary follow-up) 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 RCR monitored by DNA PCR (M12 and M24) 
 Concomitant medications 
 Adverse events 
 Disease assessments 
o 
Tumor cell assessment by flow cytometry in peripheral blood 
o 
Peripheral blood for morphologic blasts 
o 
Extramedullary disease 
o 
Response assessment by Investigator 


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 Multiple Myeloma 
Table 7-2 presents all the planned assessments and procedures for patients with MM 
6.2.1 Screening (Week -10 to Week -8) and enrollment 
In Phase I, the first three enrolled patients (regardless of indication) for each dose will be treated 
one at a time at intervals of at least 36 days. Each of these three patients will undergo 
lymphodepleting chemotherapy only once the previous patient has completed the 30-day 
follow-up or until the previous patient experiences a DLT within the completion of 30-day 
follow-up period (see section 9.1 for the definition of “evaluable” patient). The remaining 
patients for each dose will be enrolled once the dose has proven to be safe in the first three 
enrolled patients 
Any screening assessment that is done outside the screening window must be repeated prior to 
apheresis. 
Each patient will be identified by a unique patient number, assigned centrally through a web 
interface. This patient number will be used for the entire study. See the MLM-CAR44.1 Study 
Manual for details on enrollment procedures. 
 
The screening and multiple myeloma workup include the following assessments: 
 Evaluation of CD44v6 expression by tumor cells (see section below). 
 Demography (year of birth, age, sex, childbearing status of females, race). 
 Inclusion/exclusion criteria. 
 Medical history, MM diagnosis/history. 
 Prior/concomitant medications and antineoplastic therapies including surgery and 
chemo-, biologic-, immunologic- and radiation therapies. 
 International Staging System (ISS) and R-ISS (Revised-ISS): The ISS is based on 
the assessment of two blood test results, beta 2-microglobulin (ß2-M) and albumin, 
and has shown significant prognostic power for multiple myeloma. R-ISS evaluates 
the prognostic effect of adding to ISS the LDH value and the presence of high risk 
FISH results (see Appendix 2) 
 Bone marrow aspirate/biopsy for cytogenetics by means of fluorescence in situ 
hybridization (FISH). 
 Brain MRI (not required if patient has negative brain MRI within 5 weeks prior to 
screening). 
 Complete physical examination including height and weight. 
 Vital signs (temperature, heart rate, diastolic and systolic blood pressure) 
 ECOG performance status 
 DLCO or FEV1 
 Pulse oximetry 
 MUGA or ECHO 
 ECG 
 Serum pregnancy test for women of childbearing potential 


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 Local laboratory evaluations (hematology, clinical chemistry, urine analysis, 
creatinine clearance) 
 CD3/mcl count in peripheral blood 
 Coagulation panel  
 Serum β2-microglobulin and LDH 
 Viral serology (HAV, HBV, HCV, HIV, VZV, HSV 1-2, HSV-6, CMV, EBV) 
mycoplasma and toxoplasma. Not required if performed as part of clinical routine 
within 4 weeks informed consent. The details of tests requested for lymphocyte 
apheresis are specified in section 4.4 
 Absence of active infections and/or viral infections requiring the use of ganciclovir 
 Adverse events 
 Disease assessments 
o 
In serum: 
 
M-protein 
by 
protein 
electrophoresis 
(PEP) 
and 
immunofixation 
electrophoresis (IFE) 
 
Free light chain (FLC) protein assessment 
 
Corrected calcium 
o 
In urine (24-h urine collection required): 
 
M-protein by PEP and IFE; 
o 
Bone marrow aspirate/biopsy: plasma cell count, CD44v6 expression on tumor 
cells, MRD 
o 
Assessment of soft tissue plasmacytomas (STP) 
o 
Local full body skeletal survey by X-ray and/or CT/MRI 
6.2.1.1 Re-screening 
In case of screening failure, for example for an adverse event, that has delayed the execution of 
screening assessments, the investigator can re-include the patient in the clinical trial only if the 
following conditions are satisfied: 
1. 
Positivity to CD44v6 marker at first screening 
2. 
Absence of active infections and/or viral infections requiring the use of ganciclovir 
 
Re-screened patient will be identified with another screening code, but he/she will be linked 
with the previous screening code. The outcome of the re-screening phase will be considered for 
the purposes of the study. 
6.2.1.2 Evaluation of CD44v6 expression on tumor cells  
During study Phase I, CD44v6 expression on tumor cells in peripheral blood and bone marrow 
will be evaluated by a centralized laboratory at the San Raphael Hospital. The same assessment 
will also be performed at each local laboratory to set-up a validation process for CD44v6 
marking 
Patients with CD44v6 expression positive in bone marrow can be enrolled also if they have 
CD44v6 negative peripheral blood samples. 


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The threshold value of CD44v6 positivity on tumor cells should be ≥ 20%. 
In case of the result of central laboratory is different from that of local laboratory, the central 
assessment will be considered. 
Details on sample collection are provided in the MLM-CAR44.1 Study Manual. 
6.2.2 Lymphocyte apheresis (Week -9 to Week -7)  
Lymphocyte apheresis is described in Section 5.6, and full details are provided in the MLM-
CAR44.1 Study Manual. The procedure should take place no longer than 7 weeks prior to MLM-
CAR44.1 T- cell infusion and prior to the administration of chemotherapy, immunomodulators, 
or steroids to ensure an adequate ALC. 
Patients may be required to undergo multiple apheresis procedures to collect a target of 7 × 
10E8 CD3+ cells or a minimum of 5 x 10E8, or when is not possible approximately 10 x 10E9 
WBC, for the preparation of the investigational product. 
Prior to apheresis, patients should undergo a check of inclusion/exclusion criteria and 
concomitant therapies. Only after informed consent and confirmation of all eligibility criteria 
will the patient’s apheresis product be shipped to the manufacturing facility. 
6.2.3 Pre-chemotherapy evaluation visit (Week -2) 
Before lymphodepleting chemotherapy begins, the patient will undergo blood collection for 
assessments as listed below: 
 Inclusion/exclusion criteria 
 Physical examination and vital signs 
 Concomitant medications 
 Hematology, clinical chemistry 
 Bone marrow aspirate/biopsy for plasma cell count 
 Adverse events 
 Rapid influenza test (within 10 days of infusion) If the patient is positive, oseltamivir 
phosphate or zanamivir should be administered for 10 days as preventative treatment 
prior to CAR T-cell infusion 
At the discretion of the Investigator, assessments may be repeated for up to 60 days until the 
patient is considered suitable for lymphocyte depleting chemotherapy. 
6.2.4 Lymphodepleting chemotherapy (Day -5 to Day -3) 
Patients will undergo lymphodepleting chemotherapy with cyclophosphamide i.v. (500 g/m2) 
and fludarabine i.v. (30 mg/m2) daily from day -5 to day -3. 
The following assessments should be performed within 24 hours prior to starting 
lymphodepleting chemotherapy.  
 Physical examination and vital signs 
 Weight 
 Urine pregnancy test for women of childbearing potential 
 Concomitant medications 


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 Adverse events 
 Verification of criteria detailed in Section 5.8 
6.2.5 Pre-infusion visit (Day -1) 
The following exams are to be performed one day prior to MLM-CAR44.1 T-cell infusion. 
 Physical exam, vital signs and weight 
 ECOG performance status assessment 
 Hematology, clinical chemistry and coagulation panel 
 MLM-CAR44.1 T-cell infusion prerequisites (see Section 5.9) 
 Concomitant medications 
 Adverse events 
 Disease assessments 
o 
In serum: 
 
M-protein by PEP and IFE 
 
FLC protein assessment 
 
Corrected calcium 
o 
In urine (24-h urine collection required): 
 
M-protein by PEP and IFE; 
6.2.6 MLM-CAR44.1 T-cell infusion (Day 0) 
See Section 5.9 for details of MLM-CAR44.1 T-cell infusion. Patients will undergo the 
following assessments the day of infusion (but prior to infusion): 
 Physical examination, weight and vital signs pre-, during and post-infusion as 
indicated in Section 5.9.2. 
 Pulse oximetry 
 ECOG performance status 
 Hematology and clinical chemistry 
 ECG 
 MLM-CAR44.1 PK (pre-infusion) by flow cytometry in peripheral blood 
 MLM-CAR44.PK (pre-infusion) sample collection for PCR 
 Blood sample collection (pre-infusion) for ancillary studies 
 Plasma sampling (pre-infusion) for cytokine monitoring  
 Blood sample collection for RCR monitoring (pre-infusion) by DNA PCR 
 Concomitant medications 
 Adverse events 
 See Section 11.6.1 for safety monitoring guidelines following infusion 


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6.2.7 Post-infusion visits: D4±1d, D7±1d, D11±1d, D14±1d, D18±1d, D21±1d, 
D±25±1d, D28±1d (Month 1) 
Patient hospitalization with close monitoring is recommended for at least 15 days after CAR-
T-cell infusion. See Section 11.6.1 for details. 
The following assessments will be performed at the post-infusion visits: 
 Physical examination and vital signs: all visits 
 Weight: D28 
 Pulse oximetry: D4, D7, D11, D14 
 ECOG performance status: D7, D14, D21, D28 
 Hematology and clinical chemistry: all visits 
 Urine analysis: D7 and D21 
 Coagulation panel: D7, D14, D28 
 CD44v6 expression on tumor cells in peripheral blood: D7, D14, D21 and D28 
 CD44v6 expression on tumor cells in bone marrow: D28 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry: D7, D14, D21 and D28 
 MLM-CAR44.1 PK (peripheral blood) by PCR: D7, D14, D21 and D28 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry on D28 
 Blood sample collection for ancillary studies: D7, D14, D21 and D28 
 Plasma sampling for cytokine monitoring: D7, D14, D21, D28  
 Concomitant medications: all visits 
 Adverse events: all visits 
 See Section 11.6.1 for safety monitoring guidelines following infusion 
Frequent monitoring of serum CRP, ferritin and cytokines should be considered during CRS of 
any severity, especially in case of persisting fever, hemodynamic instability, respiratory distress 
and rapid clinical deterioration. See Section 11.6.1 for CRS management. 
On Day 28, assessments will include an evaluation of disease response as follows: 
 In serum: 
o 
M-protein by PEP and IFE 
o 
FLC protein assessment (if serum PEP or urine PEP or both is/are non-measurable 
and to identify sCR) 
o 
Corrected calcium 
 In urine (24-h urine collection required): 
o 
M-protein by PEP and IFE 
 Bone marrow aspirate/biopsy for plasma cell count 
 MRD in bone marrow in case of CR. 
 Response assessment by Investigator 
 Assessment of soft tissue plasmacytomas 


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6.2.8 Post-infusion visits: Weekly from W5±3d to W8±3d (Month 2) 
From Week 5 to Week 8 patients will undergo the following assessments during weekly visits: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 CD44v6 expression on tumor cells in peripheral blood (Day 60 only) 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 60 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 60 only) 
 Blood sample collection for ancillary studies (Day 60 only)  
 Plasma sampling for cytokine monitoring (Day 60 only)Concomitant medications 
 Adverse events 
On Week 8, assessments will include an evaluation of disease response as follows: 
 In serum: 
o 
M-protein by PEP and IFE 
o 
FLC protein assessment (if serum PEP or urine PEP or both is/are non-measurable 
and to identify sCR) 
o 
Corrected calcium 
 In urine (24-h urine collection required): 
o 
M-protein by PEP and IFE 
 Assessment of soft tissue plasmacytomas 
 Disease response by Investigator 
6.2.9 Post-infusion visit: W10±3d and W12±3d (Month 3) 
The following assessments will be performed on Week 10 and on Week 12: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry  
 Urine analysis (Week 10) 
 CD44v6 expression on tumor cells in peripheral blood (Day 90 only) 
 CD44v6 expression on tumor cells in bone marrow (Day 90 only) 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 90 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 90 only) 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry (Day 90 only) 
 Blood sample collection for ancillary studies (Day 90 only) 
 Plasma sampling for cytokine monitoring (Day 90 only) 
 RCR monitored by DNA PCR (Day 90 only) 
 Concomitant medications 
 Adverse events 
 Disease assessments (Week 12 only) 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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o 
In serum: 
 
M-protein by PEP and IFE 
 
FLC protein assessment (if serum PEP or urine PEP or both is/are non-
measurable and to identify sCR) 
 
Corrected calcium 
o 
In urine (24-h urine collection required): 
 
M-protein by PEP and IFE 
o 
Bone marrow aspirate/biopsy for plasma cell count  
o 
MRD in bone marrow in case of CR  
o 
Assessment of soft tissue plasmacytomas 
o 
Disease response by Investigator 
6.2.10 Post-infusion visits: Monthly from M4±14d to M6±14d 
One or more of the following assessments will be performed monthly from Month 4 to Month 
6: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 CD44v6 expression on tumor cells in peripheral blood (Day 180 only) 
 CD44v6 expression on tumor cells in bone marrow (Day 180 only) 
 MLM-CAR44.1 PK (peripheral blood) by flow cytometry (Day 180 only) 
 MLM-CAR44.1 PK (peripheral blood) by PCR (Day 180 only) 
 Blood sample collection for ancillary studies (Day 180 only) 
 Plasma sampling for cytokine monitoring (Day 180 only) 
 MLM-CAR44.1 PK (bone marrow) by flow cytometry (Day 180 only) 
 RCR monitored by DNA PCR (Day 180 only) 
 Concomitant medications 
 Adverse events 
 Disease assessments 
o 
In serum: 
 
M-protein by PEP and IFE 
 
FLC protein assessment (if serum PEP or urine PEP or both is/are non-
measurable and to identify sCR) 
 
Corrected calcium 
o 
In urine (24-h urine collection required): 
 
M-protein by PEP and IFE 
o 
Bone marrow aspirate/biopsy for plasma cell count – Month 6 only 
o 
MRD in bone marrow in case of CR at Month 6 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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o 
Assessment of soft tissue plasmacytomas 
o 
Response assessment by Investigator 
6.2.11 Post-infusion visits: M9±14d, M12±14d, M15±14d, M18±14d, M21±14d, M24 
±14d (end of primary follow-up) 
One or more of the following assessments will be performed during visits at Months 9, 12, 15, 
18, 21 and 24: 
 Physical examination, vital signs and weight 
 ECOG performance status 
 Hematology and clinical chemistry 
 CD44v6 expression on tumor cells in peripheral blood 
 RCR monitored by DNA PCR (M12 and M24) 
 Concomitant medications 
 Adverse events 
 Disease assessments 
o 
In serum: 
 
M-protein by PEP and IFE 
 
FLC protein assessment (if serum PEP or urine PEP or both is/are non-
measurable and to identify sCR) 
 
Corrected calcium 
o 
In urine (24-h urine collection required): 
 
M-protein by PEP and IFE 
o 
Assessment of soft tissue plasmacytomas 
o 
Response assessment by Investigator 
 End of primary follow-up and premature withdrawal 
Patients will be followed up for 24 months from the date of infusion. If a patient discontinues 
prematurely from the primary follow-up, a visit should be scheduled as soon as possible, at 
which time all the assessments listed for the Month 24 visit will be performed, except for RCR 
evaluation.  
Patients may voluntarily withdraw from treatment and primary follow-up at any time or be 
discontinued from the study due to reasons including: 
 Disease progression/Relapse after remission 
 Pursuing HSCT while in remission 
 Voluntary withdrawal 
Patients who discontinue or withdraw from primary follow-up early will be asked to continue 
the study in a secondary follow-up phase through Month 24. For patients who are lost to follow-
up, the investigator should make every effort to contact the patient. 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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Following completion of primary follow-up, patients will be followed for survival every 3 
months until the end of the study or will be enrolled in a long-term follow-up study, whichever 
occurs first.  
 Secondary follow-up 
Patients who discontinue primary follow-up will continue to be followed in the secondary 
follow-up phase to collect health authority requested data (e.g. protocol defined adverse events) 
up to 2 years after MLM-CAR44.1 T-cell infusion. Depending on when discontinuation occurs, 
patients will have visits at M3, M6, M9, M12, M18 and M24 as outlined in Table 7-3 and 
undergo the following assessments: 
 RCR by PCR: M3, M6, M12, M24 
 Adverse events and adverse events of special interest (including new malignancies), 
pregnancies as detailed in Section 11.4. 
 Antineoplastic therapies 
If the patient cannot attend a visit, he or she should be contacted by phone so at least survival 
status can be determined. 
 Long-term follow-up 
Patients will be followed for 15 years after MLM-CAR44.1 infusion as per health authority 
guidelines. 
Annual evaluations will be performed on all patients who have received an MLM-CAR44.1 T-
cell product infusion as recommended by the FDA and EMA and will consist in assessing 
overall health including new/recurrent malignancies, neurologic disorders, hematologic 
disorders, autoimmune disorders, unexpected medical problems and hospitalizations through a 
specific checklist. 
For the first 5 years, a blood sample for RCR testing will be collected from treated patients. In 
case the samples collected until M24 were always negative, the subsequent yearly samples will 
be taken but not analysed; in case of one or more positive samples, the culture test will be 
performed for confirmation. 
The purpose of long-term follow-up is to detect adverse reactions, manage their clinical 
consequences, and thereby collect information on the long-term safety of the treatment. For 
these reasons, the long-term follow-up should be maintained also for patients who have 
interrupted treatment and who have withdrawn from the study. A separate informed 
consent/assent form will be provided for this protocol. 
Patient alert cards that inform treating physicians about the study treatment will be provided to 
patients. These alert cards will be approved by the Ethics Committee and contain as minimum 
the name of the patient, the investigator’s contact number and information regarding the 
medical treatment received 
 Quality of Life assessment 
Not Applicable 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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 Contraception 
A woman is considered of childbearing potential, i.e. fertile, following menarche and until 
becoming post-menopausal unless permanently sterile. A postmenopausal state is defined as no 
menses for 12 months without an alternative medical cause. A high follicle stimulating hormone 
(FSH) level in the postmenopausal range may be used to confirm a post-menopausal state in 
women not using hormonal contraception or hormonal replacement therapy. However, in the 
absence of 12 months of amenorrhea, a single FSH measurement is insufficient.  
For the purpose of this document, a man is considered fertile after puberty unless permanently 
sterile by bilateral orchidectomy. 
Women of childbearing potential and all male participants must use highly effective methods 
of contraception for at least 12 months following MLM-CAR44.1 CAR T-cell infusion and 
until CAR T-cells are no longer present by flow cytometry on two consecutive tests. Highly 
effective contraception methods include: 
a) 
Total abstinence. Periodic abstinence and withdrawal are not acceptable. 
b) 
Female sterilization (hysterectomy, bilateral salpingectomy and bilateral oophorectomy)  
c) 
Male sterilization (at least 6 months prior to screening). 
d) 
BOTH of the following forms of contraception must be utilized: 
 Use of oral, injected or implanted hormonal methods of contraception or other forms 
of hormonal contraception that have comparable efficacy (failure rate <1%), for 
example hormonal vaginal ring or transdermal hormonal contraception 
 Barrier methods of contraception: condom or occlusive cap (diaphragm or 
cervical/vault caps) with spermicidal foam/gel/film/cream/vaginal suppository. 
Sexually active males must accept to use a condom during intercourse for 12 months after 
treatment. A condom is required to be used also by vasectomized men (as well as during 
intercourse with a male partner or sterile female partner) as WBCs are a normal part of semen 
and transmission of CAR T transduced cells may occur.


MolMed S.p.A.
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.2 
Confidential 
page 75 of 150 
 
7 
SCHEDULE OF ASSESSMENTS 
TABLE 7-1 
SCHEDULE OF ASSESSMENTS - ACUTE MYELOID LEUKEMIA 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival follow-up every 3 months 
 
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/month 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
Day 25 
Day 28 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 ± 14 
days 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demography 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inc./Exc. criteria 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior antineoplastic 
therapy 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AML 
diagnosis/history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bone marrow / blood: 
Morphology 
Immunophenotyping 
and if applicable 
Cytogenetics 
Fluorescence in situ 
hybridization (FISH) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brain MRI scan 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD44v6 expression 
in bone marrow and 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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page 76 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival follow-up every 3 months 
 
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/month 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
Day 25 
Day 28 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 ± 14 
days 
peripheral blood 
(central assessment) 
Safety Assessments 
Physical examination
X 
Symptom-directed physical examination 
 
Vital signs 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
 
 
X 
X 
X 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
Post-infusion safety 
monitoring 
 
 
 
 
 
See Section 11.6.1 
 
 
 
 
 
 
Pulse oximetry 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Performance status 
score (ECOG) 
X 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
Adverse events 
Continuously 
X 
Prior/concomitant 
medication, 
transfusions 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
DLCO/FEV1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serology (HAV, 
HBV, HCV, HIV, 
VZV, HSV 1-2, 
HSV-6, CMV, EBV, 
toxoplasma) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum pregnancy test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine pregnancy test 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Rapid influenza A 
and B testing 
 
 
Within 10 days of 
infusion 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MUGA/ECHO 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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page 77 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival follow-up every 3 months 
 
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/month 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
Day 25 
Day 28 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 ± 14 
days 
ECG 
X 
 
 
 
 
X 
As clinically indicated 
 
Lab Assessments 
Hematology 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
CD3/mcl count in 
peripheral blood 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical chemistry
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urine analysis 
X 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Coagulation (PT, 
aPTT, INR, 
fibrinogen, D-dimer) 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
Mycoplasma test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disease assessments 
Peripheral blood for 
morphologic blasts 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
Tumor cell 
assessment by flow 
cytometry in 
peripheral blood 
(tumor cells and 
CD44v6 assessment) 
X 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
Week 8 
 
X 
Month 6 only 
X 
 
Bone marrow 
aspirate/biopsy 
morphology and 
flow cytometry 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
 
Month 6 
 
 
MRD in bone 
marrow 
aspirate/biopsy 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X1 
Week 81 
 
 
Month 61 
 
 


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CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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page 78 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival follow-up every 3 months 
 
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/month 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
Day 25 
Day 28 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 ± 14 
days 
Extramedullary 
disease 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
Response assessment 
by Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
CD44v6 Assessment, Pharmacokinetics, safety and ancillary studies 
CRS assessments in 
peripheral blood 
(cytokines, markers) 
 
 
 
 
 
As clinically indicated 
 
CD44v6 assessment 
in bone marrow 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
 
Day 180 only 
 
 
Plasma sample for 
cytokine monitoring 
 
 
 
 
 
X 
(pre-) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 only 
 
 
MLM-CAR44.1 PK 
by flow cytometry 
and PCR (peripheral 
blood)2 
 
 
 
 
 
X 
(pre-) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 only 
 
 
MLM-CAR44.1 PK 
bone marrow by flow 
cytometry 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Day 60 
 
 
Day 180 only 
 
 
RCR monitored by 
DNA PCR 
 
 
 
 
 
X 
(pre-) 
 
 
 
 
 
 
 
 
 
 
Day 
90 
Day 180 
Month 12 and 
Month 24 
 
Sample collection for 
ancillary studies3 
 
X 
 
 
 
X 
(pre-) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 
 
 
Intervention 
MLM-CAR44.1 T-
cell infusion 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lymphocyte 
apheresis 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


MolMed S.p.A. 
CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
Confidential 
page 79 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival follow-up every 3 months 
 
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/month 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
Day 25 
Day 28 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 ± 14 
days 
Lymphodepleting 
chemotherapy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD44v6 infusion 
prerequisite 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Survival follow-up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
1 If CR/CRi is confirmed 
2 Flow cytometry to be performed both locally and at a central laboratory, PCR at a central laboratory 
3 Ancillary studies: Characterization of T-cells on Days 0, 7, 14, 21, 28, Week 8, Week 12 and Month 6 (Bambino Gesù Pediatric Hospital laboratory) 
                              CAR T immunogenicity on lymphocyte apheresis visit (Week -9 to Week -7), day 28 and Month 6 (MolMed laboratory) 
 
 
 


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CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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TABLE 7-2 
SCHEDULE OF ASSESSMENTS - MULTIPLE MYELOMA 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival every 3 
months
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/mont
h 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 ± 1d 
Day 7 ± 1d 
Day 11 ± 1d 
Day 14 ± 1d 
Day 18 ± 1d 
Day 21 ± 1d 
Day 25 ± 1d 
Day 28 ± 1d 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 (end 
of follow-
up) ± 14 
days 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demography 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inc./Exc. Criteria 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior antineoplastic 
therapy 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MM 
diagnosis/history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Internal Staging 
System (ISS and R-
ISS) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brain MRI1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cytogenetics (FISH) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD44v6 expression 
in bone marrow and 
peripheral blood 
(central assessment) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety Assessments 
Physical examination
X 
Symptom-directed physical examination 
 
Vital signs 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
 
 
X 
X 
X 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
Post-infusion safety 
monitoring 
 
 
 
 
 
See Section 11.6.1 
 
 
 
 
 
 


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CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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page 81 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival every 3 
months
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/mont
h 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 ± 1d 
Day 7 ± 1d 
Day 11 ± 1d 
Day 14 ± 1d 
Day 18 ± 1d 
Day 21 ± 1d 
Day 25 ± 1d 
Day 28 ± 1d 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 (end 
of follow-
up) ± 14 
days 
 
Pulse oximetry 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Performance status 
score (ECOG) 
X 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
Adverse events 
Continuously 
X 
Prior/concomitant 
medication, 
transfusions 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
DLCO or FEV1
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serology (HAV, 
HBV, HCV, HIV, 
VZV, HSV 1-2, 
HSV-6, CMV, EBV, 
toxoplasma) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum pregnancy test
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine pregnancy test  
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Rapid influenza A 
and B testing 
 
 
Within 10 days of 
infusion 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MUGA/ECHO 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ECG 
X 
 
 
 
 
X 
As clinically indicated 
 
Lab Assessments 
Hematology 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
CD3/mcl count in 
peripheral blood 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical chemistry
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urine analysis 
X 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Coagulation (PT, 
aPTT, INR, 
fibrinogen, D-dimer) 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 


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CLINICAL STUDY PROTOCOL 
Internal Code: IPR/33.C 
 
 
IPR Mod.3 
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page 82 of 150 
 
Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival every 3 
months
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/mont
h 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 ± 1d 
Day 7 ± 1d 
Day 11 ± 1d 
Day 14 ± 1d 
Day 18 ± 1d 
Day 21 ± 1d 
Day 25 ± 1d 
Day 28 ± 1d 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 (end 
of follow-
up) ± 14 
days 
 
Serum β2-
microglobulin, LDH 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mycoplasma test  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Efficacy Assessments 
Efficacy / Disease Assessment required to confirm response or disease progression 
SPEP, UPEP 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
Week 8  
 
X 
X 
X 
 
SIFE, UIFE 
X 
 
 
 
X 
 
. 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
To confirm CR regardless of whether measurable M-protein was present at baseline.  
 
SFLC  and UFLC 
assay 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
Analyzed only when serum M-protein or urine M-protein or both assessed by PEP is/are non-measurable and to identify sCR in case 
CR criteria are met.  
 
Plasma cell count in 
bone marrow 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
Month 6 
only 
 
 
MRD in bone 
marrow 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X2 
 
 
X2 
Month 6 
only2 
 
 
Corrected calcium 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
Skeletal survey  
X 
. 
 
 
 
As clinically indicated; CT/MRI in case of newly symptomatic areas with no X-ray finding.  
 
Assessment of 
extramedullary soft 
tissue plasmacytoma 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
Response 
assessment by 
Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Week 8 
 
X 
X 
X 
 
CD44v6 Assessment, Pharmacokinetics, safety and ancillary studies 
CRS assessments in 
peripheral blood 
(cytokines, markers) 
 
 
 
 
 
As clinically indicated 
 


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Screening 
Pre-Treatment 
Treatment and Primary Follow-Up 
Survival every 3 
months
Lymphocyte 
apheresis 
Pre-chemotherapy 
Lymphodepleting 
chemotherapy 
Pre-infusion 
Infusion 
Post-infusion 
Month 1 
Month 2 
Month 3 
 
Study 
day/week/mont
h 
W -10 to W -
8 
W -9 to W -7 
Week -2 
Day -5 to 
day -3 
Day -1 
Day 0 
Day 4 ± 1d 
Day 7 ± 1d 
Day 11 ± 1d 
Day 14 ± 1d 
Day 18 ± 1d 
Day 21 ± 1d 
Day 25 ± 1d 
Day 28 ± 1d 
Week 5 to 
Week 8  
Week 10 
Week 12 
Month 4 
to Month 
6 ± 14 
days 
Month 9, 
12, 15, 18, 
21, 24 (end 
of follow-
up) ± 14 
days 
 
CD44v6 assessment 
in peripheral blood  
X 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
Week 8 
 
X 
Day 180 
X 
 
CD44v6 assessment 
in bone marrow 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
Day 180 
 
 
Plasma sample for 
cytokine monitoring 
 
 
 
 
 
X 
(pre) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 
 
 
MLM-CAR44.1 PK 
by flow cytometry 
and PCR (peripheral 
blood)3 
 
 
 
 
 
X 
(pre) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 
 
 
MLM-CAR44.1 PK 
– bone marrow by 
flow cytometry 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Day 
90 
Day 180 
 
 
RCR monitored by 
DNA PCR 
 
 
 
 
 
X 
(pre) 
 
 
 
 
 
 
 
 
 
 
Day 
90 
Day 180 
Month 12 and 
Month 24 
 
Sample collection 
for ancillary studies4 
 
X 
 
 
 
X 
(pre) 
 
X 
 
X 
 
X 
 
X 
Day 60 
 
Day 
90 
Day 180 
 
 
Intervention 
MLM-CAR44.1 T-
cell infusion 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lymphocyte 
apheresis 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lymphodepleting 
chemotherapy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD44v6 infusion 
prerequisite 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Survival follow-up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
M protein by electrophoresis in serum (SPEP) and urine (UPEP). M protein by immunofixation in serum (SIFE) and urine (UIFE). Free light chain protein assessment (SFLC). 


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Minimal residual disease (MRD) 
1 Not required if negative MRI available within 5 weeks prior to screening. 
2 In case of CR. 
3 Flow cytometry to be performed both locally and at a central laboratory, PCR at a central laboratory 
4 Ancillary studies: Characterization of T-cells on Days 0, 7, 14, 21, 28, Week 8, Week 12 and Month 6 (Bambino Gesù Pediatric Hospital laboratory) 
                              CAR T immunogenicity on lymphocyte apheresis visit (Week -9 to Week -7), day 28 and Month 6 (MolMed laboratory) 
 
 
 
 
 
 


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TABLE 7-3 
SCHEDULE OF ASSESSMENTS – SECONDARY FOLLOW-UP (AML AND MM) 
 
Secondary follow-up 
Survival every 3 months 
after study completion 
Post-infusion 
Study month 
 
M3 ±14d 
M6±30d 
M9±30d 
M12±30d 
M18±60d 
M24±60d 
Antineoplastic medications 
 
X 
X 
X 
X 
X 
X 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
 
Pregnancies 
 
X 
X 
X 
X 
X 
X 
 
RCR by DNA PCR 
 
X 
X 
 
X 
 
X 
 
Survival follow-up 
 
 
 
 
 
 
 
X 
 
 


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8 
EVALUATION CRITERIA 
 Expression of CD44v6 on tumor cells – AML and MM patients’ study 
inclusion 
In study phase I, positivity of tumor cells in peripheral blood and bone marrow to CD44v6 will 
be evaluated by a centralized laboratory at the San Raphael Hospital during the screening phase.  
AML patients: CD44v6 positivity is required for bone marrow and peripheral blood samples; a 
positivity at least for the peripheral blood sample is required for some AML with extra-medullar 
involvement such as cutaneous localization (see M4 AML). 
MM patients: CD44v6 positivity is required for bone marrow; patients with CD44v6 expression 
positive in bone marrow can be enrolled also if they have CD44v6 negative peripheral blood 
sample. 
The threshold value of CD44v6 positivity of tumor cells should be ≥ 20%. 
This is a mandatory requirement, as CD44v6 is the target antigen of transduced MLM-CAR44.1 
T-cells. 
The same evaluation will be performed at each local laboratory. 
In case of the result of central laboratory is different from that of local laboratory, the central 
assessment will be considered. 
 Maximum tolerated dose (Phase I) - AML and MM 
The maximum tolerated dose (MTD) and recommended Phase IIa dose of MLM-CAR44.1 T-
cells will be established through a BOIN study design (see Section 3). 
 Disease response (Phase I and Phase IIa) – AML 
Response or disease progression will be assessed by the investigator based on ELN guidelines 
as indicated in Appendix 3. Response is based on bone marrow and blood morphologic criteria, 
physical examination findings, along with laboratory assessments of bone marrow minimal 
MRD. Disease assessments should be performed as reported in Table 8-1. 


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TABLE 8-1 
DISEASE ASSESSMENTS AML 
Peripheral blood for morphologic blasts 
Screening, Week -2 (pre-chemotherapy), monthly until Month 6 
and every 3 months thereafter 
Flow cytometry of peripheral blood (tumor 
cell, CD44v6 assessment) 
Screening, Days 7, 14, 21, 28 and Months 2, 3, 6, 9, 12 15, 18, 21 
and 24. 
Flow cytometry of bone marrow (CD44v6 
assessment) 
Screening, Day 28 (Month1), Week 8 (Month 2) and Month 6 
Bone marrow aspirate/biopsy for 
morphologic blast cell count/flow 
cytometry 
Screening, Week -2 (pre-chemotherapy), Day 28 (Month1), Week 
8 (Month 2) and Month 6 
MRD assessments in bone marrow by 
flow cytometry 
Screening, Day 28 (Month1), Week 8 (Month2) and Month 6 in 
case of CR/CRi 
8.3.1 Morphology 
Cell morphology will be performed according to standard procedure. Myeloblasts, monoblasts, 
and megakaryoblasts are included in the blast count. In AML with monocytic or 
myelomonocytic differentiation, monoblasts and promonocytes, but not abnormal monocytes, 
are counted as blast equivalents. Erythroblasts are not counted as blasts except in the rare 
instance of pure erythroid leukemia. 
8.3.2 Tumor cell assessment by flow cytometry  
Flow cytometry of peripheral blood (tumor cells and CD44v6 assessment) will be performed at 
screening, , Days 7, 14, 21, 28 and Months 2, 3, 6, 9, 12, 15, 18, 21 and 24. Bone marrow exams 
will be performed at screening, Day 28, Week 8 and Month 6. 
Flow cytometry provides an insight into differentiation pathways, maturation stages and 
abnormal features of the cell populations which are clinically relevant for the diagnosis of 
hematological malignancies. The presence and absence of antigens on or in the cell populations 
are recognized by various Monoclonal Antibodies (MAb) which gives characteristic 
immunostaining defining the cell lineages thus helping in making the diagnosis of acute 
leukemias. Flow cytometric immunophenotyping is a rapid reliable method not only to diagnose 
but also to assess prognosis, decision making for targeted therapy and follow up (in minimal 
residual disease evaluation) in acute leukemia (Gajendra 2016).  
Flow cytometric immunophenotyping of peripheral blood and bone marrow samples will be 
initially performed locally. 
8.3.3 Minimal residual disease (MRD) in bone marrow 
MRD in AML refers to the presence of leukemic cells below the threshold of detection using 
conventional morphologic methods. Patients with a CR according to morphologic assessment 
alone can potentially harbor a large number of leukemic cells in the bone marrow. The most 
frequently use methods for MRD assessment include multicolor flow cytometry to detect 
abnormal immunophenotypes and qPCR assays to detect NPM mutation if positive at baseline. 
For MRD evaluation with qPCR, sampling of bone marrow mononuclear cells (MNC) is 
preferred.  


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8.3.4 Extramedullary disease 
Extramedullary disease is to be assessed via physical examination, CSF assessment, and if 
required, by imaging techniques. Patients with suspected CNS leukemia must be evaluated by 
lumbar puncture and be free of CNS disease prior to study entry. Previously treated CNS 
leukemia is allowed provided adequate treatment has been given and the patient is free of CNS 
disease. 
8.3.5 Molecular cytogenetics by means of fluorescence in situ hybridization (FISH) 
Fluorescent in situ hybridization (FISH) is an option to detect gene rearrangements, such as 
RUNX1-RUNX1T1, CBFB-MYH11, MLL and EVI1 gene fusions, or loss of chromosome 5q 
and 7q material. FISH is frequently necessary to identify MLL fusion partners in 11q23 
translocations. A cytogenetic analysis of bone marrow cells will be performed at screening.  
 Disease response (Phase I and Phase IIa) MM 
Response or disease progression will be assessed by the investigator based on the analysis of 
serum and urine protein electrophoresis (SPEP and UPEP), serum and urine immunofixation 
(SIFE and UIFE), serum free light chain protein (SFLC), imaging and bone marrow 
assessments as per modified IMWG guidelines (Appendix 4). 
SPEP, UPEP, and serum calcium corrected for albumin should be collected as shown in Table 
8-2 until disease progression. After screening, IFE should be done only when endogenous M-
protein is 0 or nonquantifiable.  
TABLE 8-2 
DISEASE ASSESSMENTS MM 
M-protein by electrophoresis 
Serum (SPEP), Urine (UPEP) 
 
Screening 
Pre-infusion 
Monthly from Month 1 to Month 6, then every 3 months from 
Month 9 to Month 24. 
As needed to confirm disease progression. 
M-protein by immunofixation 
Serum1 (SIFE), Urine (UIFE) 
 
Screening 
Pre-infusion 
Monthly from Month 1 to Month 6, then every 3 months from 
Month 9 to Month 24 in subjects with disappearance of M-protein 
by PEP. 
As needed to confirm disease progression 
Free light chain  
Serum (SFLC), Urine (UFLC) 
Screening 
Pre-infusion 
Monthly from Month 1 to Month 6, then every 3 months from 
Month 9 to Month 24 if serum PEP or urine PEP or both is/are 
non-measurable. 
As needed to confirm sCR and confirm disease progression for 
subjects with serum FLC only disease. 
Clinical 
assessment 
of 
soft 
tissue 
plasmacytoma (STP) 
Screening 
Monthly from Month 1 to Month 6, then every 3 months from 
Month 9 to Month 24. 
As needed to confirm disease progression 
CT/MRI assessment of STP 
At screening and as clinically indicated.  


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Plasma cell count in bone marrow 
At screening, Week -2 (pre-chemotherapy), Day 28 (Month 1), 
Week 12 (Month 3) and Month 6 
Skeletal survey by X-ray and/or CT/MRI 
At screening and as clinically indicated, CT/MRI in case of newly 
symptomatic areas with no X-ray finding. 
Corrected calcium 
Screening 
Pre-infusion 
Monthly from Month 1 to Month 6, then every 3 months from 
Month 9 to Month 24. 
As needed to confirm disease progression 
MRD by means of RT-qPCR  
At screening, Day 28 (Month 1), Week 12 (month 3) and Month 
6 in case of CR. 
Flow cytometry of peripheral blood 
(CD44v6 assessment) 
Screening 
Days 7, 14, 21, 28 and Months 2, 3, 6, 9, 12 15, 18, 21 and 24 if 
positive at screening 
Flow cytometry of bone marrow aspirate 
(CD44v6 assessment) 
Screening, Day 28, Week 12 (Month 3) and Month 6. 
1 IFE should only be done when endogenous M-protein is 0 or non-quantifiable. 
 
Confirmation of response 
Response must be confirmed for all categories other than stable disease (SD) to rule out errors. 
A consecutive assessment can be performed at any time and confirmation should be obtained 
by M-protein assessments. 
 Bone marrow assessments do not need to be repeated, but at least 1 bone marrow 
assessment is required to substantiate a CR. 
 If imaging studies were done, they need to rule out new lytic bone lesions. 
 Should confirmation assessments reveal a better category (e.g., VGPR after PR), the 
response category of the previous assessment will be considered as confirmed (PR). 
 Should confirmation assessments reveal a worse category, (e.g., VGPR after CR), 
the response category of the subsequent assessment will be considered as confirmed 
(VGPR). 
 Should repeated measurements of a variable result in more values, the worst 
assessment is to be considered. 
Confirmation of progressive disease 
Confirmation is based on M-protein measurement or serum FLC levels. Confirmation of an 
increase in M-protein or FLC should be obtained as soon as possible. 
8.4.1 M-protein Assessment by PEP and IFE 
Response to treatment is based on M-protein levels in serum and urine. Two methods are 
required: 
 Protein electrophoresis provides quantitative measurements. 
 Immunofixation provides qualitative measurements (present/absent). It is a more 
sensitive method than PEP and is used to confirm the absence of M-protein by PEP 
(CR). 


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o 
Serum and urine IFE is required at baseline and to confirm CR regardless of whether 
measurable M-protein was present at baseline. 
o 
Subjects with measurable disease with SPEP will be assessed for response based on 
SPEP and not by the serum FLC assay. 
o 
Subjects with measurable disease in both SPEP and UPEP will be assessed for 
response based on these two tests and not by the serum FLC assay. 
Analysis by IFE will be done for all subjects at screening and baseline, and thereafter (see Table 
7-2) only in subjects with disappearance of M-protein by PEP. 
8.4.2 Free Light Chain (FLC) Protein Assessment 
The serum FLC assay measures free kappa light chain (0.33-1.94 mg/dL) and free lambda light 
chain (0.57-2.63 mg/dL). The FLC ratio is defined as the kappa serum level divided by the 
lambda serum level. Lambda is the involved light chain if FLC ratio < 0.26. Kappa is the 
involved light chain if FLC ratio is > 1.65. The FLC ratio is considered normal if FLC ratio is 
within 0.26-1.65, and abnormal if FLC ratio is < 0.26 or > 1.65. 
The kappa/lambda ratios by urine FLC immunoassays and then the cut-offs for the 
kappa/lambda ratio are defined as 5.5 for BJP K and 0.1 for BJP lambda. 
FLC will be analyzed only when serum M-protein or urine M-protein or both assessed by PEP 
is/are non-measurable (i.e., serum M-protein < 0.5 g/dL or urine M-protein < 200 mg per 24 
hours) and to identify sCR if CR criteria are met. 
8.4.3 Plasma cell count in bone marrow 
A bone marrow aspirate and biopsy for plasma cell quantification will be performed and the 
percentage of plasma cells determined through cytological/histological examination. Clonal 
plasma cell will be determined by either flow cytometry or immunohistochemistry or 
immunofluorescence, considering the following MM markers: CD45, CD14, CD2, CD19, 
CD27, CD56, CD38, CD138, CD117 and CD81. 
Either bone marrow aspirate or biopsy can be used for this assessment, but the same method 
should be used throughout the study. Bone marrow aspirate is required for MRD and flow 
cytometry assessment.  
A bone marrow aspirate and biopsy should be performed for every subject at screening, Week 
-2 (pre-chemotherapy, Day 28 (Month 1), Week 12 (month 3) and Month 6. 
8.4.4 Assessment of soft tissue plasmacytoma (STP) 
Clinical assessment 
The investigator should perform a clinical exam to assess the presence of STP at screening and 
as indicated in Table 7-2. If the presence of a STP is suspected during the study, a CT or MRI 
must be performed immediately to confirm and document the lesions’ location. 
CT/MRI  
 Screening: A CT/MRI will be performed at screening. Any imaging assessments 
already completed during the regular work-up of the subject within 40 days prior to 
lymphocyte apheresis can be considered as the baseline assessment. 


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 Post-baseline: Assessments should be performed using the same imaging technique 
used at baseline. In case of suspicion of STP and/or disease progression (based on 
clinical exam or symptoms), a CT/MRI should be performed promptly to confirm 
suspicion 
8.4.5 Skeletal survey 
A skeletal survey is to be performed by conventional radiography for osteolytic disease at 
screening. The survey will be performed during the study if clinically indicated. Use of 
conventional or low -dose CT scan or MRI is acceptable. If imaging is performed on treatment 
for assessment of progression, the same imaging technique as the one used at screening must 
be used. The number and location of skeletal lesions and whether they are lytic should be 
recorded on the eCRF. An on-treatment survey should record any increase or improvement in 
the number or size of lytic lesions. 
8.4.6 Minimal residual disease (MRD) 
MRD assessment by RT-qPCR or NGS (Next-Generation-Sequencing) is an effective tool in 
the assessment of patients with MM. MRD will be analyzed when bone marrow sample is 
obtained at screening to establish the baseline clone, and at Day 28 (Month 1), Week 12 (Month 
3) and Month 6 in case of CR. Additional assessments are to be performed 12, 18 and 24 months 
post CR/sCR in subjects who maintain CR or sCR, until PD is observed.  
8.4.7 Corrected calcium 
Corrected calcium in serum for determination of hypercalcemia as part of response assessment 
will be evaluated at screening and as indicated in Table 7-2 until disease progression using the 
following formula: 
Corrected Calcium, mg/dL = (0.8 x [normal albumin, g/dL - subject’s albumin, g/dL] + serum 
Ca, mg/dL). 
8.4.8 Cytogenetics by means of fluorescence in situ hybridization (FISH) 
A cytogenetic analysis of bone marrow cells will be performed at screening through FISH; 
should be considered at least the following abnormalities del17p, t(4;14), t(14,16). The 
pathological bone marrow cells from patients with MM are characterized by genetic instability 
resulting in numerical and structural chromosomal abnormalities that may serve as prognostic 
factors. 
8.4.9 CD44v6 evaluation by flow cytometry 
CD44v6 evaluation by flow cytometry of peripheral blood will be performed at screening and 
on Days 7, 14, 21, 28 and Months 2, 3, 6, 9, 12, 15, 18, 21 and 24 if positive at screening. 
Bone marrow exams will be performed at screening, Day 28, Week 12 (Month 3) and Month 
6. 
 Safety assessments (AML and MM) 
See Section 11.6.1 for safety monitoring guidelines following MLM-CAR44.1 T-cell infusion. 


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8.5.1 Adverse Events and safety monitoring 
The overall safety of treatment with MLM-CAR44.1 T-cells will be evaluated by analyzing the 
type, frequency and severity of adverse events (AE) and by monitoring for systemic reactions 
(fever, tachycardia, nausea and vomiting, joint pain, skin rash) for 30 days following CAR T-
cell infusion. See Section 11.4 for details about adverse events and Section 11.6 for guidelines 
about safety monitoring. 
8.5.2 Physical examination 
Physical examinations will be performed daily until Day 15 and then at every scheduled visit. 
A full physical examination will be performed at screening, whereas targeted exams will occur 
during the treatment and follow-up periods according to the investigator’s observations and 
complaints from the patient. 
Significant findings that were present prior to the signing of informed consent must be included 
in the Medical History eCRF page. Significant new findings that begin or worsen after informed 
consent must be recorded on the Adverse Event page of the eCRF. 
8.5.3 Eastern Cooperative Oncology Group (ECOG) Performance Status 
ECOG Performance Status score will be evaluated using the criteria described in Appendix 6. 
The assessment should be completed prior to any study-related procedures or assessments. 
8.5.4 Vital signs 
Vital signs include temperature, blood pressure, pulse and respiratory rate. 
8.5.5 Height and weight 
Height in centimeters (cm) and body weight (to the nearest 0.1 kilogram in indoor clothing, but 
without shoes). 
8.5.6 Cardiac assessments 
An ECG will be performed at screening, day 0 (prior to infusion) and throughout the study as 
clinically indicated. An ECHO/MUGA test is required to be completed at screening. Clinically 
significant abnormalities present when the patient signed the informed consent should be 
reported on the Medical History eCRF page. New or worsened clinically significant findings 
occurring after informed consent must be recorded on the Adverse Events CRF page. Patients 
must have a left ventricular ejection fraction (LVEF) >45% to be included into the study. 
8.5.7 DLCO or FEV1 
Carbon monoxide diffusing capacity (DLCO) or forced expiratory volume in one second 
(FEV1) will be assessed at screening. 
8.5.8 Pregnancy test 
Women of childbearing potential will be required to undergo a serum pregnancy test at 
screening and a urine pregnancy test prior to lymphodepleting chemotherapy. 
8.5.9 Replication-competent retrovirus (RCR) testing 
Although extremely unlikely, an RCR may be generated during CAR-T-cell manufacturing or 
following infusion. The development of RCR could represent a risk to both the patient and their 


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close contacts, and therefore monitoring for RCR will take place during the trial by means of 
DNA PCR. If a positive RCR assay is obtained from a blood specimen, the patient will be 
rescheduled for a retest. Should the test be confirmed, public health officials are to be informed 
and the patient should be followed up intensively together with gene therapy experts, study 
investigators and Health Authorities. 
 Laboratory tests 
Laboratory assessments will be performed according to Table 8.3. Additional assessments 
should be performed between visits as clinically indicated and to follow adverse events or 
events related to treatment with MLM-CAR44.1 T-cells. See Section 11.6.1 for safety 
monitoring guidelines following MLM-CAR44.1 T-cell infusion. 
TABLE 8-3 
LOCAL CLINICAL LABORATORY ASSESSMENTS 
Hematology 
Hematocrit, hemoglobin, MCHC, MCV, platelets, red blood cells, white blood cells 
with complete differential, flow cytometry panel 
CD44v6 expression  
 
Chemistry 
Glucose, blood urea nitrogen (BUN), creatinine, creatinine clearance (at screening 
only), sodium, potassium, calcium, total protein, albumin, total bilirubin, alkaline 
phosphatase, ALT (SGPT), AST (SGOT), magnesium, phosphorus, LDH, ferritin, uric 
acid and C-reactive protein (CRP), Serum β2-microglobulin, IgA, IgG, IgM 
Urinalysis 
Macroscopic panel (dipstick) (bilirubin, blood, glucose, ketones, leukocytes esterase, 
nitrite, Ph, protein, specific gravity). 
If macroscopic panel is abnormal then perform microscopic panel (red blood cells, 
white blood cells, casts, crystals, bacteria, epithelial cells) 
Coagulation panel 
Prothrombin time (PT) or International normalized ratio (INR), activated partial 
thromboplastin time (aPTT), fibrinogen, and D-dimer 
Pregnancy screen 
Serum or urine tests 
Influenza 
Rapid Influenza A & B Test 
Serology 
HAV, HBV, HCV, HIV, VZV, HSV 1-2, HSV-6, CMV, EBV, toxoplasma, treponema 
pallidum and mycoplasma 
MLM-CAR44.1 T-
cell PK 
Flow cytometry for CAR detection in peripheral blood 
MLM-CAR44.1 T-
cell PK 
Flow cytometry for CAR detection in bone marrow at disease evaluation 
 
TABLE 8-4 
CENTRAL LABORATORY ASSESSMENTS 
CD44 v6 expression 
 
MLM-CAR44.1 T-
cell PK 
Flow cytometry and PCR of peripheral blood** 
 
Cytokine monitoring 
Multiplex technology of cytokine plasma levels** 
Assessments for 
ancillary studies 
Functional and phenotypic characterization of T cells** 
CAR T immunogenicity* 
Replication 
competent retrovirus 
(RCR) 
PCR in peripheral blood* 
*Studies performed at MolMed laboratory 
**Studies performed at Ospedale Pediatrico Bambino Gesù laboratory 
 


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 Pharmacokinetics (AML and MM) 
Blood sampling for local PK evaluations by flow cytometry will be performed during the 
infusion visit (Day 0) prior to infusion, on Days 7, 14, 21, 28, 60, 90 and 180. Additional 
unscheduled samples may be collected as needed depending on the clinical course of CRS. An 
unscheduled PK sample should be collected in case of relapse. 
Bone marrow sampling for PK will take place on Day 28, 60, and 180 in AML patients and on 
Day 28, 90 and 180 in MM patients.  
Samples will also be properly stored and sent to a central laboratory, where peripheral blood, 
and plasma samples will be analyzed through flow cytometry, qPCR and multiplex cytokine 
technology. 
 Ancillary studies 
In addition to assessing the efficacy and safety of MLM-CAR44.1 T-cells, the study aims to 
perform exploratory assessments that address specific features and concerns related to CAR T-
cell therapy. 
We aim to characterize the cellular composition of circulating LNGFR+ (i.e. CAR-T cells) and 
LNGFR- T cells in patients on Days 0, 7, 14, 21, 28, 60, 90 and 180. Characterization will 
include, but is not limited to, the presence of naïve (CD45RA+/CD95-/CD62L+), stem memory 
(CD45RA+/CD95+/CD62L+), central memory (CD45RA-/CD62L+), effector memory 
(CD45RA-/CD62L-), terminally differentiated (CD45RA+/CD62L) as well as T regulatory 
cells. Moreover, the expression of activation and exhaustion markers will be assessed.   
If sufficient biological material is available on the sampling days mentioned above, and if more 
than 10% of LNGFR+ T cells are present in the patient’s biological material, functional 
analyses will be performed including: 
1) Activation by challenging with CD44v6+ and CD44v6- AML and MM tumor cell lines. 
In this case the readout will be specific T cell proliferation and/or cytokine production.  
2) By-standard activation of tumor specific T cells recognizing tumor-specific peptides 
(i.e. WT1, PR1 and NYESO1 pepmix). In this case the readout will be specific T cell 
cytokine production. 
To this aim, frozen PBMC collected at the time points defined above will be shipped to a central 
laboratory. A priority level has been set in case the recovered PBMC are not sufficient to 
perform all the proposed analysis. 
The immunogenicity of the CAR polypeptide as well as against suicide genes has been 
described in several studies. If an immune response to MLM-CAR44.1 T-cells occurs, the cells 
might be rejected. To monitor T cell-mediated immune responses against CAR CD44v6 or TK 
products, PBMC will be collected at lymphocytes apheresis, d28, d180, or after CAR T-cell 
disappearance, and shipped to a central laboratory. Samples will be analyzed in case of CAR 
T-cells disappearance: they will be stimulated in vitro in the presence of irradiated autologous 
CAR T cells and tested against target cells expressing TKmut2 and /or CAR CD44v6.  
Additional samples from peripheral blood and/or bone marrow may be collected after MLM-
CAR44.1 T cell infusion according to medical and scientific judgement (i.e. during CRS or 
other clinically relevant event).  


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If the patient agrees, the remaining biomarker and/or PK samples collected as well as any 
patient’s baseline lymphocyte apheresis product that is not used for manufacturing and any 
MLM-CAR44.1 manufactured product that is not infused will be stored for up to 15 years and 
further analyzed to address scientific questions related to MLM-CAR44.1 or cancer, including 
research related to improvements in the manufacturing process. The assessments will include 
but are not limited to immune-related DNA, RNA, or protein markers, qPCR, and/or gene 
expression profiling. A decision to perform such additional research studies would be based on 
outcome data from this study or from new scientific findings related to the drug class or disease, 
as well as on reagent and assay availability. 
 
 


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9 
STATISTICAL CONSIDERATIONS 
 Statistical design 
This is a seamless Phase I/IIa, open label, multicenter clinical trial that combines a Phase I dose-
escalation component based on toxicity with a Phase IIa dose expansion based on anti-tumor 
activity for AML and MM populations separately. 
Phase I: To select the Maximum Tolerated Dose (MTD) from the three specified doses, we 
assumed a maximum sample size of 15 patients (in AML and MM separately) treated in cohorts 
of three and a target toxicity rate of 30%.  
A patient is defined “evaluable” if, after CAR-T cell infusion, can be included in one of the 
following cases: 
 The patient completes the 30 days of safety follow-up period with no occurrence of 
DLTs;  
 The patient experiences a DLT within the completion of 30-day follow-up period; 
 The patient dies before the completion of the 30-day follow-up period with no previous 
occurrence of DLTs. In this case, the patient is considered as having experienced a DLT. 
A patient is defined “not evaluable” and should be replaced during the study if one of the 
following conditions occurs before the completion of the 30-day follow-up period: 
 The patient receives a rescue medication (i.e.: Ganciclovir or Valganciclovir 
administered for Grade 2 or higher bullous dermatitis; Tocilizumab/Siltuximab 
administered for Grade 3 or higher CRS responsive to therapy within 24 hours OR for 
Grade 2 or lower CRS OR for Grade 2 or lower CRES) without previous occurrence of 
DLTs; 
 The patient prematurely discontinues the study (alive) without the occurrence of DLTs. 
Phase IIa: A total of 14 patients with each indication will be treated at the MTD (AML and MM 
separately) according to a Simon’s two-stage design, considering alpha (type 1 error) = 0.01, 
power = 0.90 and assuming p0=0.10 and p1=0.50, being p0 the probability of hematological 
disease response in case of a poorly effective drug and p1 the desired probability required to 
proceed to further development. 
9.1.1 Primary study variable 
Phase I 
 DLT 
The proportion of patients with DLT occurring within one month from CAR T infusion will be 
calculated for each dose level along with the 95% exact Clopper-Pearson confidence intervals. 
 Overall safety: type and frequency of SAEs, of treatment-emergent AEs and of 
systemic reactions 
SAEs, treatment-emergent AEs and systemic reactions (i.e. fever, tachycardia, nausea and 
vomiting, joint pain, skin rash) will be summarized for each dose level by system organ class 
and/or preferred term and severity by means of frequency tables. The description will be done 
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1) Pre-infusion period (from the day of apheresis to the day before CAR T-cell infusion 
2) Post-infusion period (30 days following CAR T-cell infusion). 
 Absence of replication competent retrovirus 
The proportion of patients with absence of replication competent retrovirus monitored by DNA 
PCR at 3, 6, 12 and 24 months after infusion of MLM-CAR44.1 T-cells will be calculated for 
each dose level. The corresponding 95% exact Clopper-Pearson confidence intervals will also 
be calculated. 
Phase IIa 
 Hematologic disease response for AML at 2 months 
The percentages of patients either in CR, Cri or PR (ELN criteria) at 2 months following MLM-
CAR44.1 T-cell infusion will be estimated, and the 95% exact Clopper-Pearson confidence 
intervals calculated. 
 Hematologic disease response for MM at 3 months 
The overall response rate (ORR) is defined as the proportion of patients in: stringent complete 
response (sCR), CR, very good partial response (VGPR) and partial response (PR) (IMWG 
criteria). The percentages of overall response at 3 months following MLM-CAR44.1 T-cell 
infusion will be estimated, and the 95% exact Clopper-Pearson confidence intervals calculated. 
9.1.2 Secondary/exploratory study variables 
Phase I 
 Hematologic disease response for AML 
The percentages of patients either in CR, CRi or PR (ELN criteria) at 1 and 2 months following 
MLM-CAR44.1 T-cell infusion will be estimated at each dose level and the 95% exact Clopper-
Pearson confidence intervals calculated. 
 Hematologic disease response for MM 
The overall response rate (ORR) is defined as the proportion of patients in: stringent complete 
response (sCR), CR, very good partial response (VGPR) and partial response (PR) (IMWG 
criteria). The percentages of overall response at 1 and 3 months following MLM-CAR44.1 T-
cell infusion will be estimated at each dose level and the 95% exact Clopper-Pearson confidence 
intervals calculated. 
 Suicide gene activation  
The percentage of patients for whom the activation of the suicide gene was needed will be 
calculated for each dose level along with the corresponding 95% exact Clopper-Pearson 
confidence intervals. 
The levels of transduced cells in patients presenting CRS or other significant MLM-CAR44.1 
T-cell related toxicities and treated with ganciclovir will be listed and described at each dose 
level with measures of location (i.e., mean, median) and variability (i.e., standard deviation, 
interquartile range, range), as appropriate.  
 In vivo pharmacokinetic profile  


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Details of the pharmacokinetic analysis will be provided in the statistical analysis plan. 
 To set-up a validation process among the participating centers for the CD44v6 marking 
of tumor cells (AML and MM).  
Details of the analysis will be provided in the statistical analysis plan. 
 Ancillary study 
Details of the ancillary study will be provided in the statistical analysis plan 
Phase IIa 
Efficacy 
 Hematologic disease response for AML 
The percentages of patients either in CR, CRi or PR (ELN criteria) at 1 and 6 months after 
MLM-CAR44.1 T-cell infusion and those of patients in morphologic leukemia-free state 
(MLFS) 1, 2 and 6 months after infusion will be estimated and the 95% exact Clopper-Pearson 
confidence intervals calculated. 
 Hematologic disease response for MM 
ORR at 1, 2 and 6 months following MLM-CAR44.1 T-cell infusion will be estimated, and the 
95% exact Clopper-Pearson confidence intervals calculated. 
 Overall Survival (OS) 
Overall Survival (OS) is defined as the time from the date of MLM-CAR44.1 T-cell infusion 
to the date of last follow-up or death due to any cause, whichever occurs first.  
Censoring may be due to: 
- study closure 
- lost to follow-up 
- withdrawn consent 
A description of the overall survival time will be performed using median and range. The 
Overall Survival distribution function will be estimated at 2 years using the Kaplan-Meier 
method on the Full Analysis Set and 95% CI will also be calculated.  An additional analysis of 
OS will be performed not censoring at the new anticancer therapy (including >HSCT), so that 
these patients should be followed-up for survival. 
 Disease-Free Survival (DFS) in AML patients 
Disease Free Survival (DFS) is the time from achievement of response to the earliest of the 
following: lost follow-up, relapse or death due to any cause.  
Censoring may be due to: 
- study closure 
- lost to follow-up 
- withdrawn consent 
- new anticancer therapy (including HSCT) 
A description of the DFS will be performed using median and range. The DFS distribution 
function will be estimated at 2 years using the Kaplan-Meier method on the Full Analysis Set 


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and 95% CI will also be calculated. Additionally, a sensitivity analysis of DFS will be 
performed without censoring at SCT. 
 Progression Free Survival (PFS) in MM patients 
Progression Free Survival (PFS) is the time from the date of MLM-CAR44.1 T-cell infusion to 
the earliest of the following: last follow-up, progression/relapse or death due to any cause.  
Censoring may be due to: 
- study closure 
- lost to follow-up 
- withdrawn consent.  
- new anticancer therapy (including HSCT) 
A description of the PFS will be performed using median and range. The PFS distribution 
function will be estimated at 2 years using the Kaplan-Meier method and 95% CI will also be 
calculated. Additionally, a sensitivity analysis of PFS will be performed without censoring at 
SCT. 
 Duration of response (DOR) in MM patients 
Duration of response (DOR) is defined as the time from achievement of response to the date of 
last follow-up, relapse or death due to any cause, whichever occurs first.  
Censoring may be due to: 
- study closure 
- lost to follow-up 
- withdrawn consent.  
- new anticancer therapy (including HSCT) 
A description of the DOR will be performed using median and range. The DOR distribution 
function will be estimated using the Kaplan-Meier method and 95% CI will also be calculated. 
Additionally, a sensitivity analysis of DOR will be performed without censoring at SCT. 
 Event-Free Survival (EFS) in AML patients 
Event Free Survival (EFS) is the time from the date of MLM-CAR44.1 T-cell infusion to the 
date of the earliest of the following: last follow-up, resistance, relapse or death due to any cause.  
Censoring may be due to: 
- study closure 
- lost to follow-up 
- withdrawn consent 
- new anticancer therapy (including HSCT) 
A description of the EFS will be performed using median and range. The EFS distribution 
function will be estimated at 2 years using the Kaplan-Meier method on the Full Analysis Set 
and 95% CI will also be calculated. Additionally, a sensitivity analysis of EFS will be 
performed without censoring at SCT. 
 Minimal Residual Disease for AML 
The percentages of patients with a molecular CR at 2 months following MLM-CAR44.1 T-cell 
infusion will be estimated, and the 95% exact Clopper-Pearson confidence intervals calculated. 


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 Minimal Residual Disease for MM 
The percentages of patients with a molecular CR at 3 months following MLM-CAR44.1 T-cell 
infusion will be estimated, and the 95% exact Clopper-Pearson confidence intervals calculated. 
Feasibility 
 Percentage of eligible patients who undergo both lymphocyte apheresis and CAR T-cell 
infusion 
The percentages of eligible patients who undergo both lymphocyte apheresis and CAR T-cell 
infusion will be estimated and the 95% exact Clopper-Pearson confidence intervals calculated 
 Percentage of eligible patients effectively infused with CAR T-cells after undergoing 
lymphocyte apheresis. 
The percentages of eligible patients effectively infused with CAR T-cells after undergoing 
lymphocyte apheresis will be estimated and the 95% exact Clopper-Pearson confidence 
intervals calculated.  
Safety 
 Overall safety: type and frequency of SAEs, treatment-emergent AEs and systemic 
reactions 
SAEs, treatment-emergent AEs and systemic reactions (i.e. fever, tachycardia, nausea and 
vomiting, joint pain, skin rash) will be summarized for each dose level by system organ class 
and/or preferred term and severity by means of frequency tables. The description will be done 
for 1) the pre-infusion period (from the day of apheresis to the day before CAR T-cell infusion) 
and 2) for the post-infusion period (30 days following CAR T-cell infusion). 
 Absence of replication competent retrovirus 
The proportion of patients with absence of replication competent retrovirus monitored by DNA 
PCR at 3, 6 and 12 and 24 months after infusion of MLM-CAR44.1 T-cells will be calculated. 
The corresponding 95% exact Clopper-Pearson confidence intervals calculated. 
 Absence of abnormal MLM-CAR44.1 T-cell clonal expansion 
The proportion of patients with absence of abnormal MLM-CAR44.1 T-cell clonal expansion 
monitored by clinical and laboratory surveillance, as well as by TCR-Vbeta repertoire study 
will be calculated 6 months after infusion. The corresponding 95% exact Clopper-Pearson 
confidence intervals will also be calculated. 
 Suicide gene activation  
The percentage of patients for whom activation of suicide gene was needed will be calculated 
along with the corresponding 95% exact Clopper-Pearson confidence intervals. 
The levels of transduced cells in patients presenting CRS or other significant MLM-CAR44.1 
T-cell related toxicities and treated with ganciclovir will be listed and described with measures 
of location (i.e., mean, median) and variability (i.e., standard deviation, interquartile range, 
range), as appropriate.  
Pharmacokinetic profile 


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Details of the pharmacokinetic analysis will be provided in the statistical analysis plan. 
Ancillary study 
Details of the ancillary study will be provided in the statistical analysis plan 
9.1.3 Analysis populations 
The analysis sets are shown in the Table below. The analyses will be generally performed 
separately on AML and MM, unless otherwise specified. The safety and the efficacy analyses 
will be conducted on the Full Analysis Set.  
 
Analysis set 
Description 
Full Analysis Set 
(Safety Set) 
All screened patients who have received infusion of MLM-CAR44.1 T-
cells 
Per-Protocol Set 
Subset of the patients in the Full Analysis Set who are compliant with 
major requirements of the clinical study protocol. Major protocol 
deviations leading to exclusion from the Full Analysis Set are: 
- patients who receive a dose which differs for more than 10% of the target 
dose (±10%). 
9.1.4 Data Quality Assurance 
Data collection 
Designated investigator staff will enter the data required by the protocol into the eCRF using 
fully validated software that conforms to 21 CFR Part 11 requirements. Designated investigator 
site staff will not be given access to the Electronic Data Capture system until they are trained.  
Web-based software will be used, and no installation procedure is needed. Each site will be 
authorized by the administrator to access the eCRF. Each site-qualified personnel will be 
allowed to access the eCRF by means of a ‘login mask’ requiring user ID and password and 
may read, modify, and update only the information entered at his or her site and according to 
their profile. Each page reports site code and subject code. 
On-line validation programs will check for data discrepancies and, by generating appropriate 
error messages, allow the data to be confirmed or corrected before transfer to the CRO working 
on behalf of the Sponsor. The investigator will certify that the data entered in the eCRF are 
complete and accurate.  
After database lock, the investigator will receive a CD-ROM of subject data for archiving at 
the investigational site. 
Database management and quality control 
The CRO working on behalf of the Sponsor will review the data entered in the eCRF by 
investigational staff for completeness and accuracy and instruct site personnel to make any 
necessary corrections or additions. The Data Manager will perform the cleaning session by 
reviewing the warning messages raised by on-line checks and by running post-entry checks by 
means of validation programs and data listings specific for the study. The occurrence of any 
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If clarifications are needed, the Data Manager will raise queries through the web application. 
Designated investigator site staff will be required to respond to queries and the Data Manager 
will make the correction to the database according to the responses. 
Data collection and query flows, as well as the on-line and off-line checks, are detailed in the 
Data Management Plan and Data Validation documents. 
Concomitant medications and prior medications entered into the database will be coded using 
the WHO Drug Reference List, which employs the ATC classification system. Medical 
history/current medical conditions and AEs will be coded using MedDRA. 
The database will be locked after all the above actions have been completed and the database 
has been declared complete and accurate. 
 Sample size 
Phase I 
No formal sample size calculation was performed for this study as no inference is foreseen. 
However, simulation study for the dose escalation process was done to assess the performances 
of the BOIN design for different sample sizes. 
Phase IIa 
A total of 14 patients will be treated at the MTD for each indication (AML and MM separately) 
according to a Simon’s two-stage design, considering alpha (type 1 error) = 0.01, power = 0.90 
and assuming p0=0.10 and p1=0.50, being p0 the probability of hematological disease response 
in case of a poorly effective drug and p1 the desired probability required to proceed to further 
development. 
 
 
 


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10 
DATA AND SAFETY MONITORING BOARD (DSMB)  
Safety oversight will be under the direction of a Data and Safety Monitoring Board (DSMB).  
 DSMB roles and responsibilities 
This DSMB will 
 Review the Statistical Analysis Plan for DSMB 
 Meet periodically to review aggregate and individual subject data related to safety, 
data integrity and overall conduct of the trial. 
 Provide recommendations to continue or terminate the trial depending upon these 
analyses.   
 Communicate other recommendations or concerns as appropriate. 
 Suggest/request additional analyses (if necessary) 
 Operate according to the procedures described in the DSMB Charter and all 
regulatory guidelines in place.    
 Follow conflict of interest guidelines as detailed in the DSMB Charter. 
 Comply with confidentiality procedures as described in the DSMB Charter. 
 Maintain documentation and records of all activities as in the DSMB Charter. 
 Composition of the DSMB 
During Phase I, the DSMB will be made up of a Hematologist with expertise in CAR T-cell 
therapy, a Biostatistician and a Dermatopathologist collectively have expertise in the 
management of patients with the study indication, experience in Phase I clinical trials, statistical 
methods and safety monitoring. A Pediatrician will join the DSMB during phase IIa 
 
 


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 Organizational diagram and data flow 
 
 
 
 
 
DSMB 
Review of documents and 
statistical reports. 
Voting and making 
recommendations
CRO Data Management 
Data cleaning and extraction of 
data for DSMB meeting 
Investigational Sites 
Data entry onto eCRF  
CRO Biostatistician 
Production of statistical report 
for DSMB 
(Tables, Figures and Listings) 
MOLMED & EURE-CAR T Steering 
Committee 
Review and implement DSMB 
recommendations 
MolMed 


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 DSMB governance 
The DSMB will operate under the rules of an approved charter and its members will be 
independent from study conduct and free of conflict of interest.  
The initial meeting of the DSMB will be organizational and will be held after the DSMB group 
has been established to let the members review and approve the DSMB charter, to establish a 
meeting schedule, review the study modifications and/or termination guidelines and discuss the 
format of the open and closed reports that will be used to present trial results at future DSMB 
meetings 
 
 


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11 
SAFETY 
 Definitions 
An Adverse Event (AE) is any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not necessarily 
have a casual relationship with this treatment. An adverse event (AE) can therefore be any 
unfavourable and unintended sign (including an abnormal laboratory finding, for example), 
symptom, or disease temporally associated with the use of a medicinal product, whether or not 
considered related to the medicinal product. 
An Adverse Drug Reaction (ADR) is any untoward and unintended response to an 
investigational medicinal product related to any dose administered. 
An Unexpected Adverse Drug Reaction is any adverse reaction, the nature or severity of 
which is not consistent with the applicable product information (e.g. Investigator’s Brochure). 
A Serious Adverse Event (SAE) or a Serious Adverse Drug Reaction (SADR) is any 
untoward medical occurrence that at any dose: 
 results in death  
 is life-threatening  
 results in persistent or significant disability or incapacity 
 is a congenital anomaly or birth defect  
 is medically significant 
 requires inpatient hospitalization or prolongation of existing hospitalization 
 hospitalizations for the following reasons should not be reported as SAEs: 
o 
elective or pre-planned treatment for a pre-existing condition that is unrelated to the 
indication under study and has not worsened since signing the informed consent 
o 
treatment on an emergency outpatient basis that does not result in hospital 
admission and involves an event not fulfilling any of the definitions of a SAE given 
above  
Medical and scientific judgment should be exercised in deciding whether expedited reporting 
is appropriate in other situations, such as important medical events that may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the patient or may 
require intervention to prevent one of the outcomes listed in the definitions above. These 
situations should also usually be considered Serious Adverse Events. 
Any clinically significant laboratory test value that meets the definition of a SAE must be 
reported as a SAE. 
 Criteria of evaluation 
All adverse events will be recorded on the case report forms (CRFs) according to CTC version 
5.0 except for CRS and CRES, which will follow table 11-2. The investigator will decide if the 
events are drug related and the decision will be recorded for all adverse events.  


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The adverse events not listed on the NCI CTC grading system will be graded on a five points 
scale and reported in detail on the CRF, as indicated: 
Mild 
Discomfort noticed but no disruption of normal daily activity 
Moderate 
Discomfort sufficient to reduce or affect daily activity 
Severe 
Inability to work or perform normal daily activity 
Life-threatening 
Represents an immediate threat of life 
Death 
Death related to the adverse event 
 
Relationship of the adverse event to the treatment should also be assessed using the following 
categories for determining relationship: 
 
Not related 
The adverse event is clearly not related to the 
investigational product  
Related to MLM-CAR44.1 T-
cells 
The adverse event is considered related to the investigational 
product. 
Related to lymphocyte 
apheresis 
The adverse event is considered related to the lymphocyte 
apheresis. 
Related to lymphodepleting 
chemotherapy 
The adverse event is considered related to the chemotherapy. 
Unknown 
This category should be used when it is difficult to determine if 
the adverse event is drug related or not, or information about it is 
unavailable, missing or incomplete. 
ALL SAEs and AEs are collected regardless of their relationship with the experimental product.  
Laboratory abnormalities that constitute an AE (are considered clinically significant, induce 
clinical signs or symptoms, require concomitant therapy or require changes in study treatment), 
should be recorded on the Adverse Events CRF. Whenever possible, a diagnosis, rather than a 
symptom should be provided (e.g. anemia instead of low hemoglobin). Laboratory 
abnormalities that meet the criteria for Adverse Events should be followed until they have 
returned to normal or an adequate explanation of the abnormality is found.  
  Adverse events of special interest (AESI) 
The following adverse events are defined as being of special interest: all AE related to study 
procedures, cytokine release syndrome, any cutaneous toxicity, tumor lysis syndrome, 
neutropenic fever, cytopenia >28 days, neurotoxicity, any fever/infection with monocytopenia 
and hepatic events. 
 Reporting procedures 
11.4.1 Adverse Events 
Adverse events that begin or worsen after informed consent/assent will be recorded in the 
patient’s source documents. New or worsening adverse events prior to starting study treatment 
(lymphocyte apheresis) are to be recorded in the Adverse Events CRF if they meet one of the 
following criteria: 


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 All infections 
 All clinical AEs grade ≥ 3 
 All clinically significant laboratory abnormalities 
 All AEs related to a study procedure 
 All AEs leading to study discontinuation 
 All SAEs. 
Once the patient begins lymphocyte apheresis, all new or worsening adverse events 
regardless of causality will be recorded in the Adverse Events CRF up to the Month 12 visit. 
Events occurring during salvage chemotherapy but prior to CAR T-cell infusion will however 
be excluded from the adverse event reporting period, except for SAE. 
Following the Month 12 visit, and through the Month 24 visit, adverse events should only be 
reported and recorded in the Adverse Events CRF if they meet one of the following criteria: 
 SAEs 
 Events leading to death 
 Events related to a study procedure 
 Infections: serious or opportunistic infections 
 New incidence or exacerbation of a pre-existing neurologic disorder 
 New incidence or exacerbation of a prior rheumatologic or other autoimmune 
disorder 
 New incidence of other hematologic disorder 
 Any condition suspected of being related to MLM-CAR44.1 T-cells. 
 Positive RCR test result 
 Vector insertion site sequencing result with a mono-or oligoclonality pattern or in a 
location near a known human oncogene 
 New malignancy other than the primary malignancy 
 Progressive multifocal leukoencephalopathy (PML) 
 Hepatitis B reactivation 
11.4.2 Serious Adverse Events 
Every SAE, regardless of suspected causality, occurring after signature of informed consent to 
Month 24 must be reported to within 24 hours of learning of its occurrence and be inserted in 
CRF. 
The follow-up information of SAE should describe whether the event has resolved or continues, 
if and how it was treated, and whether the patient continued or withdrew from study 
participation. 
Information about all SAEs will be recorded using the safety tool integrated into the eCRF. 
In case of technical difficulties, SAE notification can be carried out by contacting the Contract 
Research Organization (CRO) in charge of Pharmacovigilance via email at all_phv@opis.it or 
by fax using the following number: Fax: +39 0362 633622; Tel: +39 0362 311, +39 349 
3925306  


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The event is nevertheless to be reported on the eCRF once technical difficulties have been 
resolved. 
As this is a “first in man” study, Serious Adverse Drug Reactions (SADR) are not present in 
the reference safety information of the Investigator’s brochure. All events are therefore to be 
considered “unexpected” for reporting purposes. 
11.4.3 DLTs 
All DLTs must be reported within 24 hours of learning of their occurrence and be inserted in 
the specific page of the eCRF 
 Procedures to be followed in the event of pregnancy 
The investigator shall report all pregnancy exposure occurring in a female patient or in a male 
patient’s partner within 24 hours to the Sponsor using the safety tool integrated into the eCRF. 
In case of technical difficulties, pregnancy notification can be carried out by contacting the 
Contract Research Organization (CRO) OPIS in charge of Pharmacovigilance via email at 
all_phv@opis.it or by fax using the following number: Fax: +39 0362 633622; Tel: +39 0362 
311, +39 349 3925306 
The investigator should counsel the subject; discuss the risks of continuing with the pregnancy 
and the possible effects on the fetus. Monitoring of the subject should continue until conclusion 
of the pregnancy 
 Risk management plan 
11.6.1 Safety monitoring 
The two most commonly observed toxicities with CAR T-cell therapies are: 
 cytokine-release syndrome (CRS), characterized by high fever, hypotension, 
hypoxia, and/or multiorgan toxicity;  
 CAR T-cell related encephalopathy syndrome (CRES), characterized by a toxic 
encephalopathic state with symptoms of confusion and delirium, and occasionally 
seizures and cerebral edema (Neelapu et al., 2017; Lee et al., 2014; Brudno et al., 
2016; Maude et al., 2014).  
Rare cases of fulminant macrophage-activation syndrome (MAS) characterized by severe 
immune activation, lymphohistiocytic tissue infiltration, and immune-mediated multiorgan 
failure, have also been reported. 
11.6.1.1 Patient monitoring after CAR-T-cell infusion 
Patient hospitalization with close monitoring is recommended for at least 15 days after CAR-
T-cell infusion. Monitoring should include assessment of vital signs at least every 4 h, and daily 
review of organ systems, physical exam, complete blood count with differential, complete 
metabolic profile, coagulation profiles, and measurement of serum CRP and ferritin levels (see 
Tables below). Laboratory tests, including blood counts and chemistry panel, might need to be 
performed more than once daily, especially for patients at high risk of severe CRS and/or CRES, 
or those with a high tumor burden, who are at risk of tumor lysis; for the latter group, 


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precautions to avoid tumor lysis should be used, as per standard institutional guidelines. Owing 
to a high risk of arrhythmias, cardiac monitoring by telemetry is advised from the time of CAR-
T-cell infusion until resolution of any emergent CRS symptoms. Additional investigations, such 
as chest radiography, electrocardiography, echocardiography, electroencephalography (EEG), 
and imaging studies, can be performed as needed, depending on the toxicities that arise. Daily 
fluid balance and bodyweights should be strictly monitored, and maintenance of intravenous 
hydration is recommended for all patients with, or at risk of developing CRS. Central venous 
access is recommended, preferably with a double or triple lumen catheter, before CAR-T-cell 
infusion, to facilitate the timely delivery of any medications needed to manage toxicities. 
Packed red blood cells and platelets can be transfused according to standard institutional 
guidelines. Corticosteroids should be avoided for the management of fever or for premedication 
before blood transfusions to avoid limiting the effectiveness of CAR-T-cell therapy, but 
growth-factor support with filgrastim can be provided for neutropenia. Patients who develop 
fever should be assessed for infection using blood and urine cultures, chest radiography, and 
additional tests, such as cytomegalovirus PCR, respiratory viral screening, and CT of the chest, 
as indicated. conditional orders for fever and hypotension should be put in place for all patients 
receiving CAR-T-cell infusion, in order that appropriately trained nursing staff can act quickly 
in the event of toxicities, thereby minimizing delays in intervention (Neelapu et al., 2017). 
TABLE 11-1 
SUPPORTIVE-CARE CONSIDERATIONS FOR CAR-T-CELL THERAPY 
Before and during CAR-T-cell infusion 
• Baseline brain MRI to rule out any central nervous system (CNS) disease. 
• Central venous access, preferably with double or triple lumen catheter, for intravenous fluid 
and other infusions in case of toxicities. 
• Cardiac monitoring by telemetry starting on the day of CAR-T-cell infusion and continued 
until cytokine-release syndrome (CRS) resolves to detect arrhythmias. 
• Tumor lysis precautions for patients with bulky tumors, as per standard institutional 
guidelines. 
• Hospitalization recommended for at least 15 days after CAR-T-cell therapy 
Patient monitoring after CAR-T-cell infusion 
 Day 1-15 
• Assess vital signs every 4 h, close monitoring of oral and intravenous fluid input and urine 
output, and daily measurement of bodyweight. 
• Daily review of patient history and physical examination. 
• Daily blood counts, complete metabolic profiling, and coagulation profiling. 
• C-reactive protein and ferritin levels measured daily, starting on the day of infusion. 
• Assessment and grading of CRS should be done at least twice daily, and whenever the 
patient’s status changes 
• Assessment and grading of CRES using the CAR-T-cell-therapy-associated toxicity 10-
point neurological assessment (CARTOX-10; Appendix 7) should be done at least every 8 h.
• Maintenance intravenous fluids with normal saline to ensure adequate hydration. 
 Day 15-30 
• Assess vital signs every 4 h, close monitoring of oral and intravenous fluid input and urine 
output, and daily measurement of bodyweight. 
• Daily review of patient history and physical examination. 
• Daily blood counts,  
• Complete metabolic profiling, coagulation profiling, C-reactive protein and ferritin levels 
measured three times a week. 


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• Assessment and grading of CRS should be done at least daily, and whenever the patient’s 
status changes 
• Assessment and grading of CRES using the CAR-T-cell-therapy-associated toxicity 10-
point neurological assessment (CARTOX-10; Appendix 7) should be done daily. 
Notifications and contingency orders 
• The physician should be notified on detection of any of the following: systolic blood 
pressure (SBP) >140 mmHg or <90 mmHg; heart rate >120 bpm or <60 bpm, or arrhythmia; 
respiratory rate >25 breaths per min or <12 breaths per min; arterial oxygen saturation <92% 
on room air; urine output <1,500 ml per day; upward trend in blood creatinine levels or the 
results of liver function tests; tremors or jerky movements in extremities; change in mental 
status (alertness, orientation, speech, ability to write a sentence, or CARTOX-10 score). 
• For patients with a temperature ≥38.3 °C, order blood cultures (central and peripheral), 
urinalysis and urine cultures, portable chest radiography, and notify physician. 
• For patients with neutropenia and fever, start empiric broad-spectrum antibiotics. 
• Corticosteroids should not be administered unless approved by physician. 
• If patient develops CRES, withhold oral intake of food, fluids, and medicine, and notify 
physician. 
11.6.1.2 Cytokine release syndrome (CRS) 
CRS is the most common toxicity of cellular immunotherapy and is triggered by the activation 
of T cells on engagement with antigens expressed by tumor cells. The activated T cells release 
cytokines and chemokines, as do bystander immune cells such as monocytes and/or 
macrophages. CRS typically presents with constitutional symptoms, such as fever, malaise, 
anorexia, and myalgias, but can affect any organ system in the body, including cardiovascular, 
respiratory, integumentary, gastrointestinal, hepatic, renal, hematological, and nervous 
systems. Patients at high risk of severe CRS include those with bulky disease, comorbidities, 
and those who develop early onset CRS within 3 days of cell infusion. The onset of CRS toxicity 
usually occurs within the first week after CART-cell therapy, and typically peaks within 1–2 
weeks of cell administration. See Section 1.4 for more details. 
CRS should be suspected if at least one of the following four symptoms or signs is present 
within the first 3 weeks of cellular immunotherapy: fever ≥ 38° C; hypotension with systolic 
blood pressure < 90 mmHg; hypoxia with an arterial oxygen saturation of < 90% on room air; 
and/or evidence of organ toxicity. 
This study will adopt the CRS grading system and treatment recommendations proposed by 
Neelapu et al. and the CAR T-cell therapy associated TOXicity (CARTOX) Working Group 
(Neelapu et al., 2017), which incorporate and expand on the criteria proposed previously by 
Lee et al. (Lee et al., 2014). The grading system is shown in the Table below: 
 
 


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TABLE 11-2 
GRADING OF CYTOKINE-RELEASE SYNDROME (CRS) 
Symptom or sign of CRS 
CRS 
grade 
1* 
CRS grade 2‡ 
CRS grade 3‡ 
CRS grade 4‡ 
Vital signs 
Temperature ≥38 °C (fever) 
Yes 
Any 
Any 
Any 
Systolic blood pressure <90 mmHg 
(hypotension) 
No 
Responds to IV fluids 
or low-dose 
vasopressors 
Needs high-dose or 
multiple 
vasopressors§ 
Life-threatening 
Needing oxygen for SaO2 >90% 
(hypoxia) 
No 
FiO2 <40% 
FiO2 ≥40% 
Needing ventilator 
support 
Organ toxicities|| 
Cardiac: tachycardia, arrhythmias, 
heart block, low ejection fraction 
• Respiratory: tachypnea, pleural 
effusion, pulmonary edema 
• GI: nausea, vomiting, diarrhea 
• Hepatic: increased serum ALT, 
AST, or bilirubin levels 
• Renal: acute kidney injury 
(increased serum creatinine levels), 
decreased urine output 
• Dermatological: rash (less 
common) 
• Coagulopathy: disseminated 
intravascular coagulation (less 
common) 
Grade 
1 
Grade 2 
Grade 3 or grade 4 
transaminitis 
Grade 4 except 
grade 4 
transaminitis 
1. 
Adapted from Lee et al. The CRS grade should be determined at least twice a day, and whenever a change in the 
patient's status is observed. ALT, alanine aminotransferase; AST, aspartate aminotransferase; FiO2, fraction of 
inspired oxygen; GI, gastrointestinal; IV, intravenous; SaO2, arterial oxygen saturation. 
2. 
*Grade 1 CRS can manifest as fever and/or grade 1 organ toxicity. 
3. 
‡For grades 2, 3, or 4 CRS, any one of the criteria other than fever is sufficient. 
4. 
§High-dose vasopressors are defined as any of the following (as reported by Lee et al.): noradrenaline ≥20 μg/min; 
dopamine ≥10 μg/kg/min; phenylephrine ≥200 μg/min; adrenaline ≥10 μg/min; if on vasopressin, vasopressin plus 
noradrenaline equivalent of ≥10 μg/min; and if on combination vasopressors (not including vasopressin), 
noradrenaline equivalent of ≥20 μg/min. The noradrenaline equivalent dose is calculated using the VASST trial 
vasopressor equivalent equation: [noradrenaline (μg/minute)] + [dopamine (μg/kg/minute)/2] + [adrenaline 
(μg/minute)] + [phenylephrine (μg/minute)/10]. 
5. 
||Grading of organ toxicities is performed according to Common Terminology Criteria for Adverse Events. 
Supportive care and anti-cytokine therapy have been used for effective management of CRS. 
Prompt responses to tocilizumab have been seen in most patients. Those with a suboptimal 
response to the first dose of tocilizumab may receive a second or third dose. If the patient 
experiences ongoing CRS despite administration of repeated doses of tocilizumab, activation 
of the MLM-CAR44.1 T-cell suicide gene through administration of ganciclovir (GCV) may 
be considered. 
A treatment algorithm for the management of CRS is presented in Table 11-3 and should be 
followed by investigators. The algorithm is designed to avoid life-threatening toxicities, while 
allowing the CAR T-cells to proliferate. 
 
 


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TABLE 11-3 
CRS MANAGEMENT ALGORITHM 
CRS grade  
Symptom or sign  
Management 
Grade 1  
Fever 
or 
organ 
toxicity  
Acetaminophen and hypothermia blanket for the treatment of fever  
Ibuprofen can be used as second treatment option for fever, if not 
contraindicated  
Assess for infection using blood and urine cultures, and chest 
radiography  
Empiric broad-spectrum antibiotics and filgrastim if neutropenic  
Maintenance intravenous (IV) fluids for hydration  
Symptomatic management of constitutional symptoms and organ 
toxicities. 
Consider tocilizumab 8 mg/kg* IV or siltuximab 11 mg/kg IV for 
persistent (lasting >3 days) and refractory fever. 
Grade 2  
Hypotension  
IV fluid bolus of 500–1,000 ml of normal saline.  
Can give a second IV fluid bolus if systolic blood pressure (SBP) 
remains <90 mmHg.  
Tocilizumab 8 mg/kg* IV or siltuximab 11 mg/kg IV for the 
treatment of hypotension that is refractory to fluid boluses; 
tocilizumab can be repeated after 6 h if needed. 
If hypotension persists after two fluid boluses and anti-IL-6 therapy, 
start vasopressors, consider transfer to intensive-care unit (ICU), 
obtain echocardiogram, and initiate other methods of hemodynamic 
monitoring  
In patients at high-risk‡ or if hypotension persists after 1–2 doses of 
anti-IL-6 therapy, dexamethasone can be used at 10 mg IV every 6 h. 
Manage fever and constitutional symptoms as in grade 1.  
Hypoxia  
Supplemental oxygen. 
Tocilizumab or siltuximab ± corticosteroids and supportive care, as 
recommended for the management of hypotension.  
Organ toxicity  
Symptomatic management of organ toxicities, as per standard 
guidelines.  
Tocilizumab or siltuximab ± corticosteroids and supportive care, as 
indicated for hypotension.  
Grade 3  
Hypotension  
IV fluid boluses as needed, as recommended for the treatment of 
grade 2 CRS.  
Tocilizumab and siltuximab as recommended for grade 2 CRS, if not 
administered previously.  
Vasopressors as needed.  
Transfer to ICU, obtain echocardiogram, and perform hemodynamic 
monitoring as in the management of grade 2 CRS.  
Dexamethasone 10 mg IV every 6 h; if refractory, increase to 20 mg 
IV every 6 h.  
Manage fever and constitutional symptoms as indicated for grade 1 
CRS. 
Activation of suicide gene with ganciclovir should be considered in 
case of Grade 3 CRS that does not respond to steroids after 
tocilizumab administration within 24 hours. 
Hypoxia  
Supplemental oxygen including high-flow oxygen delivery and non-
invasive positive pressure ventilation.  
Tocilizumab or siltuximab plus corticosteroids and supportive care, 
as described above.  
Activation of suicide gene with ganciclovir as described above. 


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CRS grade  
Symptom or sign  
Management 
Organ toxicity  
Symptomatic management of organ toxicities as per standard 
guidelines. 
Tocilizumab or siltuximab plus corticosteroids and supportive care, 
as described above.  
Activation of suicide gene with ganciclovir as described above. 
Grade 4  
Hypotension  
IV fluids, anti-IL-6 therapy, vasopressors, and hemodynamic 
monitoring as defined for the management of grade 3 CRS.  
Methylprednisolone 1 g/day IV.  
Manage fever and constitutional symptoms as in grade 1 CRS.  
Activate suicide gene with ganciclovir. 
Hypoxia  
Mechanical ventilation.  
Tocilizumab or siltuximab plus corticosteroids and supportive care, 
as described above.  
Activate suicide gene with ganciclovir. 
Organ toxicity  
Symptomatic management of organ toxicities as per standard 
guidelines.  
Tocilizumab or siltuximab plus corticosteroids and supportive care, 
as described above.  
Activate suicide gene with ganciclovir. 
Neelapu et al. 2017. 
All medication doses indicated are for adults. *Maximum amount of tocilizumab per dose is 800 mg. ‡High-risk 
patients include those with bulky disease, those with comorbidities, and those who develop early onset CRS within 
3 days of CAR-T-cell infusion. 
 
Patients with grade 3 or 4 CRS should be treated in the ICU to enable continuous monitoring, 
management of life-threatening arrhythmias, hemodynamic shock, non-invasive positive 
pressure ventilation, mechanical ventilation, and/or dialysis. Both anti-IL-6 therapy and 
corticosteroids should be used for the management of grades 3 and 4 CRS, and the associated 
organ toxicities. Suicide gene activation should be considered in case of Grade 4 CRS and 
Grade 3 CRS that does not respond to steroids after tocilizumab administration within 24 hours. 
Serum CRP levels are a useful marker to monitor in patients undergoing cellular 
immunotherapy because IL-6 induces the production of CRP by hepatocytes. Thus, an increase 
in serum CRP level is typically detected after the onset of CRS and correlates with increased 
levels of IL-6. Moreover, the return of CRP levels to baseline indicates that the CRS phase of 
CAR-T-cell therapy has ended, and the patient can be considered for discharge from the 
hospital, assuming other toxicities that require monitoring and/or intervention have resolved. 
Of note, the correlation between CRP levels and CRS is variable, and is not observed in all 
patients. 
11.6.1.3 CAR T-cell related encephalopathy syndrome (CRES) 
The earliest signs of CRES are diminished attention, language disturbance, and impaired 
handwriting; other symptoms and signs include confusion, disorientation, agitation, aphasia, 
somnolence, and tremors. In severe cases of CRES (grade >2), seizures, motor weakness, 
incontinence, mental obtundation, increased intracranial pressure, papilloedema, and cerebral 
oedema can also occur. The manifestation of CRES can be biphasic, with the first phase 


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occurring concurrently with high fever and other CRS symptoms within the first 5 days after 
CAR T-cell therapy, and the second after the fever and other CRS symptoms subside, often 
beyond 5 days after cell infusion. See Section 1.4 for more details. CRES typically lasts for 2–
4 days but can vary in duration from a few hours to weeks.  
Like CRS, the management of CRES is based on the toxicity grade (CARTOX-10 – see 
Appendix 7). Grade 1 CRES are managed primarily through supportive care. The head of the 
patient's bed should be elevated by at least 30 degrees to minimize aspiration risks and to 
improve cerebral venous flow. A neurology consultation should be requested for thorough 
neurological evaluation, including EEG and fundoscopic examination to rule out papilloedema. 
Neuroimaging and CSF opening pressure, if available, are much better surrogates of increased 
intracranial pressure and possible cerebral edema than papilloedema; however, lumbar puncture 
might also be infeasible when patients are restless or have coagulopathy. In patients with an 
ommaya reservoir, opening pressure can be measured in the supine position with the base of 
the manometer placed at heart level. Combinations of these techniques should be considered to 
diagnose increased intracranial pressure and cerebral edema. In particular, repeated neuro 
imaging, preferably with review of the results by a neuro radiologist, is recommended to detect 
early signs of cerebral edema in patients with grade 3 or 4 CRES, and in patients with rapid 
changes in the CRES grade (increase in grade by two levels, for example, grade 1 CRES 
worsening to grade 3). The clinical status of the patient often dictates the choice of 
neuroimaging modality: MRI of the brain is preferred, but cannot be performed for unstable or 
agitated patients, whereas CT can be. The development of cerebral edema in patients treated 
with CAR T cells is associated with other acute and clinically significant neurological changes, 
such as a low CARTOX-10 score and/or seizures. 
Anti-IL-6 therapy is recommended for patients with CRES and concurrent CRS. If not 
associated with CRS, corticosteroids are the preferred treatment and can be tapered after 
improvement. The optimal duration of corticosteroid therapy remains unknown, although short 
courses of steroids have been associated with resolution of neurological toxicities without 
impaired antitumor responses. Patients should be monitored closely for recurrence of 
neurotoxicity symptoms during corticosteroid tapering. Monitoring in the ICU is recommended 
for patients with grade 3 CRES and is required for all patients with grade 4 CRES because they 
might need mechanical ventilation for airway protection. Activation of suicide gene with 
ganciclovir should be considered in grade 3 or higher neurotoxicity. Non-convulsive and 
convulsive status epilepticus should be managed with benzodiazepines and additional 
antiepileptics (preferably with levetiracetam), as needed. The response of some patients to 
benzodiazepine is rapid, with improvements in both EEG findings and mental status. After 
levetiracetam, phenobarbital is the preferred second antiepileptic for the management of CRES-
related seizures: phenytoin and lacosamide are associated with higher risks of cardiovascular 
adverse effects, therefore, their use in patients with concurrent CRS should be excluded to avoid 
arrhythmias and hypotension. CRES with raised intracranial pressure should be managed 
promptly with corticosteroids and acetazolamide; patients who develop grade 4 CRES with 
cerebral oedema should receive high-dose corticosteroids, hyperventilation, and hyperosmolar 
therapy (Neelapu et al., 2017). 
 
 


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TABLE 11-4 
RECOMMENDATIONS FOR MANAGEMENT OF CAR-T-CELL CRES 
Grade 1 
• Vigilant supportive care; aspiration precautions; intravenous (IV) hydration 
• Withhold oral intake of food, medicines, and fluids, and assess swallowing 
• Convert all oral medications and/or nutrition to IV if swallowing is impaired 
• Avoid medications that cause central nervous system depression 
• Low doses of lorazepam (0.25–0.5 mg IV every 8 h) or haloperidol (0.5 mg IV every 6 h) 
can be used, with careful monitoring, for agitated patients 
• Neurology consultation 
• Fundoscopic exam to assess for papilloedema 
• MRI of the brain with and without contrast; diagnostic lumbar puncture with measurement 
of opening pressure; MRI spine if the patient has focal peripheral neurological deficits; CT 
scan of the brain can be performed if MRI of the brain is not feasible 
• Daily 30 min electroencephalogram (EEG) until toxicity symptoms resolve; if no seizures 
are detected on EEG, continue levetiracetam 750 mg every 12 h 
• If EEG shows non-convulsive status epilepticus, treat as per algorithm in BOX 3 
• Consider anti-IL-6 therapy with tocilizumab 8 mg/kg* IV or siltuximab 11 mg/kg IV, if 
CRES is associated with concurrent cytokine-release syndrome (CRS) 
Grade 2 
• Supportive care and neurological work-up as described for grade 1 CRES 
• Tocilizumab 8 mg/kg* IV or siltuximab 11 mg/kg IV if associated with concurrent CRS 
• Dexamethasone 10 mg IV every 6 h or methylprednisolone 1 mg/kg IV every 12 h if 
refractory to anti-IL-6 therapy, or for CRES without concurrent CRS 
• Consider transferring patient to intensive-care unit (ICU) if CRES associated with grade ≥2 
CRS 
Grade 3 
• Supportive care and neurological work-up as indicated for grade 1 CRES 
• ICU transfer is recommended 
• Anti-IL-6 therapy if associated with concurrent CRS, as described for grade 2 CRES and if 
not administered previously 
• Corticosteroids as outlined for grade 2 CRES if symptoms worsen despite anti-IL-6 therapy, 
or for CRES without concurrent CRS; continue corticosteroids until improvement to grade 1 
CRES and then taper 
• Activation of suicide gene with ganciclovir may be considered 
• Consider repeat neuroimaging (CT or MRI) every 2–3 days if patient has persistent grade 
≥3 CRES 
Grade 4 
• Supportive care and neurological work-up as outlined for grade 1 CRES 
• ICU monitoring; consider mechanical ventilation for airway protection 
• Anti-IL-6 therapy and repeat neuroimaging as described for grade 3 CRES 
• High-dose corticosteroids continued until improvement to grade 1 CRES and then taper; for 
example, methylprednisolone IV 1 g/day for 3 days, followed by rapid taper at 250 mg every 
12 h for 2 days, 125 mg every 12 h for 2 days, and 60 mg every 12 h for 2 days 
• Activation of suicide gene with ganciclovir 
 
All medication doses indicated are for adults. CAR, chimeric antigen receptor. *Maximum 
amount of tocilizumab per dose is 800 mg 


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11.6.1.4 On-target/off tumor recognition 
CD44v6 has a low level of expression on normal cells, including activated T cells, monocytes, 
and keratinocytes (see Section 1.4.2.2). Nevertheless, given the potential for off-tumor toxicity 
of CD44v6 CAR T-cells, patients will be closely monitored for skin toxicities and monocyte 
levels.  
In case of skin rash, it is strongly recommended to perform: 
 a dermatological visit to evaluate the possibility of drug reaction 
 a dermatological diagnosis by a specialist to identified: 
o specific skin reaction compatible with drug reaction (erythema multiforme, 
DRESS syndrome, acute generalized exanthematous pustulosis, Stevens-
Johnson Syndrome, …) → optional but recommended biopsy  
o nonspecific skin reaction (e.g. esantematic reaction), but compatible with drug 
reaction → recommended biopsy  
o skin reaction not compatible with drug reaction → if specific diagnosis, optional 
biopsy; if uncertain diagnosis, recommended biopsy. 
Details on biopsy are provided in the MLM-CAR44.1 Study Manual. 
Antihistamines and topical or systemic cortisone-based drugs should be used for treatments. In 
case of specific or controversial diagnosis, therapy should be decided by a specialist 
dermatologist. 
A DLT for Phase I of this study is the presence of: NCI-CTC grade 3 or higher erythroderma 
(generalized exfoliative dermatitis), Stevens-Johnson Syndrome (SJS), Toxic epidermal 
Necrolysis (TEN), and any other grade 3 or higher skin toxicity suspected of being related to 
CAR T-cell treatment. 
The following off-tumor skin toxicities may set off suicide gene activation through 
administration of ganciclovir 10 mg/kg/day divided into 2 administrations, or valganciclovir 
900 mg twice per day orally for 14 days: 
 Grade 3 erythroderma not responsive to steroids (methylprednisolone 2mg/kg for 72 
hours or rapidly evolving) 
 Grade 2 or higher bullous dermatitis 
 Any grade Stevens Johnson SyndromeAny evidence for Toxic Epidermal Necrolysis 
(TEN) 
Patients have to be monitored for SJS and TEN before and after chemotherapy and CAR T cell 
infusion.  
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous 
hypersensitivity reactions. The exact mechanism of Stevens-Johnson syndrome and toxic 
epidermal necrolysis is unknown; however, one theory holds that altered drug metabolism (eg, 
failure to clear reactive metabolites) in some patients triggers a T-cell-mediated cytotoxic 
reaction to drug antigens in keratinocytes. CD8+ T cells have been identified as important 
mediators of blister formation: SJS/TEN, from an immunologic standpoint, appears to behave 
most like a delayed-type hypersensitivity reaction (DTH) [9].  


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The most common drug causes include: 
 Sulfa drugs (eg, cotrimoxazole, sulfasalazine) 
 Other antibiotics (eg, aminopenicillins [usually ampicillin or amoxicillin], 
fluoroquinolones, cephalosporins) 
 Antiepileptics (eg, phenytoin, carbamazepine, phenobarbital, valproate, lamotrigine) 
 Miscellaneous individual drugs (eg, piroxicam, allopurinol, chlormezanone) 
 Cases that are not caused by drugs are attributed to: 
 Infection (mostly with Mycoplasma pneumoniae) 
 Vaccination 
 Graft-vs-host disease 
Within 1 to 3 week after the start of the offending drug, patients develop a prodrome of malaise, 
fever, headache, cough, and keratoconjunctivitis. Macules, often in a target configuration, then 
appear suddenly, usually on the face, neck, and upper trunk. These macules simultaneously 
appear elsewhere on the body, coalesce into large flaccid bullae, and slough over a period of 1 
to 3 days. Nails and eyebrows may be lost along with epithelium. The palms and soles may be 
involved. Skin, mucosal, and eye pain are common. In some cases, diffuse erythema is the first 
skin abnormality of toxic epidermal necrolysis. 
In severe cases of toxic epidermal necrolysis, large sheets of epithelium slide off the entire body 
at pressure points (Nikolsky sign), exposing weepy, painful, and erythematous skin. Painful 
oral crusts and erosions, keratoconjunctivitis, and genital problems (eg, urethritis, phimosis, 
vaginal synechiae) accompany skin sloughing in up to 90% of cases. Bronchial epithelium may 
also slough, causing cough, dyspnea, pneumonia, pulmonary edema, and hypoxemia. 
Glomerulonephritis and hepatitis may develop. 
Mortality can be as high as 7.5% in children and 20 to 25% in adults but tends to be lower with 
early treatment. 
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are clinically similar 
except for their distribution. By one commonly accepted definition, changes affect < 10% of 
body surface area in SJS and > 30% of body surface area in TEN; involvement of 15 to 30% of 
body surface area is considered SJS/TEN overlap. 
Diagnosis is often obvious from appearance of lesions and rapid progression of symptoms. 
Histologic examination of sloughed skin shows necrotic epithelium, a distinguishing feature. 
The severity-of-illness score for TEN (see Table: Severity-of-Illness Score for Toxic Epidermal 
Necrolysis (SCORTEN)) systematically scores 7 independent risk factors within the first 24 h 
of presentation to the hospital to determine the mortality rate for a particular patient. 
 
 


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TABLE 11-5 
SEVERITY-OF-ILLNESS SCORE FOR TOXIC EPIDERMAL NECROLYSIS (SCORTEN) 
Risk* Factor   
Score 0 
Score 1 
Age   
< 40 yr 
≥ 40 yr 
Associated Cancer 
No  
Yes  
Heart rate (beats/min)  
<120 
≥ 120 
Serum BUN, mg/dL (mmol/L) 
≤ 28 (10) 
> 28 (10)  
Serum bicarbonate, mEq/L (mmol/L) 
≥ 20 (20) 
<20 (20) 
Serum glucose, mg/dL (mmol/L) 
≤ 252 (14) 
>252 (14) 
*More risk factors indicate a higher score and a higher mortality rate (%) as follows: 
0–1 = 3.2% (CI: 0.1 to 16.7) 
2 = 12.1% (CI: 5.4 to 22.5) 
3 = 35.3% (CI: 19.8 to 53.5) 
4 = 58.3% (CI: 36.6 to 77.9) 
≥ 5 => 90% (CI: 55.5 to 99.8) 
CI = confidence interval. 
Data from Bastuji-Garin S, Fouchard N, Bertocchi M, et al: SCORTEN: A severity-of-illness score for toxic epidermal 
necrolysis. Journal of Investigative Dermatology 115:149–153, 2000. 
 
Cyclosporine, plasma exchange or IVIG (Intravenous immunoglobulin) and early pulse 
corticosteroid therapy have been used for treatments. 
Potentially causative drugs should be stopped immediately. Patients are isolated to minimize 
exposure to infection and are given fluids, electrolytes, blood products, and nutritional 
supplements as needed. Skin care includes prompt treatment of secondary bacterial infections 
and daily wound care as for severe burns. Prophylactic systemic antibiotics are controversial 
and often avoided. 
Ganciclovir may be administered to set off suicide gene activation in the presence of the 
following off-tumor skin toxicities: 
 Grade 3 erythroderma not responsive to steroids (methylprednisolone 2mg/kg for 72 
hours or rapidly evolving) 
 Grade 2 or higher bullous dermatitis 
 Any grade Stevens-Johnson Syndrome 
 Any evidence for Toxic Epidermal Necrolysis 
Concerning the potential on-target/off-tumor risk of monocytopenia, the following measures 
of close patient monitoring have been implemented: 
 Close monitoring of the haematological parameters, including the measurement of 
complete blood count with differentia including monocytes, at each clinical study 
visit 
 Precautionary hospitalization in a hematology ward, during the first 15 days after 
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 Twice-weekly measurement of monocytes during the first month after CAR-T 
dosing, followed by weekly measurement for the second month, every two weeks for 
the third month, monthly until Month 6 and then every 3 months until Month 24 
Patients who remain monocytopenic after MLM-CAR44.1 T cell therapy should be considered 
immune-depressed. Suicide gene activation may thus also be considered in case of grade 3-4 
potentially life-threatening infections. 
11.6.1.5 Tumor lysis syndrome (TLS) 
Patients are to be monitored for TLS before and after chemotherapy and CAR T-cell infusion. 
Prophylactic allopurinol, or a non-allopurinol alternative (e.g. febuxostat), and increased 
oral/IV hydration should be given prior to lymphodepleting chemotherapy and CAR T-cell 
infusion in patients with elevated uric acid or high tumor burden. Supportive care (i.v. hydration 
and rasburicase) should be implemented promptly if uric acid continues to rise despite 
allopurinol/febuxostat and fluids. 
Patients should be monitored frequently from the start of lymphodepleting chemotherapy for 
potassium, phosphorus, calcium, creatinine and uric acid. 
Laboratory TLS is defined as two or more of the following values within three days before or 
in the days following MLM-CAR44.1 T-cell infusion: 
 Uric acid ≥ 8 mg/dL or 25% increase from baseline 
 Potassium ≥ 6 mEq/L or 25% increase from baseline 
 Phosphorus ≥ 4.5 mg/dL or 25% increase from baseline 
 Calcium ≤ 7 mg/dL or 25% decrease from baseline 
In case of laboratory TLS, patients should be managed with i.v. fluids, laboratory blood tests 
every 6 to 8 hours and inpatient care. Cardiac monitoring should be considered, and rasburicase 
should be considered if uric acid levels remain elevated. If zero or one of the laboratory values 
are abnormal, continue to manage with allopurinol or a non-allopurinol alternative (e.g. 
febuxostat) and oral hydration. IV fluids and rasburicase should be considered if uric acid levels 
remain elevated.  
Clinical TLS is defined as the presence of laboratory TLS plus ≥ 1 of these criteria in the 
absence of other causes. 
 Serum creatinine ≥ 1.5 times the upper limit of normal range 
 Symptomatic hypocalcemia 
 Cardiac arrhythmia 
If Clinical TLS exists, manage with IV fluids, laboratory blood tests every 6 to 8 hours, cardiac 
monitoring, rasburicase/allopurinol/febuxostat and inpatient care (consider ICU). (Cairo et al, 
2004). 
11.6.1.6 Infections 
Patients who develop fever should be assessed for infection using blood and urine cultures, 
chest radiography, and additional tests, such as cytomegalovirus PCR, respiratory viral 
screening, and CT of the chest, as indicated. Such tests should also be performed before 


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initiation of CAR-T-cell therapy when infection is suspected. Therapy with CAR T-cells should 
be delayed until infection has been controlled or ruled out as undiagnosed infections can have 
catastrophic consequences in patients with CRS, probably owing to exacerbation of systemic 
inflammation associated with the underlying infection. Empiric broad-spectrum antibiotic 
therapy, including Gram-negative bacterial coverage, should be implemented because sepsis 
and CRS have overlapping symptoms and the absence of positive cultures cannot rule out 
pathogenic infection in immunocompromised patients with cancer (Neelapu et al., 2017). 
Patients who remain monocytopenic after MLM-CAR44.1 T-cell therapy should be considered 
immune-depressed. Suicide gene activation should thus also be considered in case of grade 3-
4 potentially life-threatening infections. 
11.6.1.7 Uncontrolled T-cell proliferation 
MLM-CAR44.1 T-cells could theoretically proliferate without control of normal homeostatic 
mechanisms. In pre-clinical studies and clinical experience (Grupp et al 2013, Maude et al 
2014), CTL019 transduced cells have only proliferated in response to physiologic signals or 
upon exposure to CD19 antigen. In the context of MLM-CAR44.1 T-cell therapy, it is expected 
that the T cells will proliferate in response to signals from the CD44v6 expressing malignant 
tumor and normal monocytes. If uncontrolled T-cell proliferation occurs (e.g. expansion in the 
absence of CD44v6), patients may be treated with corticosteroids such as methylprednisolone 
(2 mg/kg/d i.v.), chemotherapy, such as high dose cyclophosphamide or ganciclovir to activate 
the suicide gene. 
11.6.1.8 Replication-competent retrovirus (RCR) testing. 
A review of 29 clinical trials using gene-modified T cell products in HIV and cancer patients 
that were manufactured with gamma-retroviral vectors reported no RCR in T cell products 
tested. In addition, in the 629 follow-up patient samples from these trials, ranging from 1 month 
to 8 years, no RCR were detected (Bear et al 2012). The absence of replication-competent RCR 
will be monitored in this study by DNA PCR for the Galv and gag-pol genes. 
11.6.2 Urgent Safety Measures (USM) 
To reduce any extra risks that may arise from multicenter clinical trial, appropriate measures 
have been taken to ensure the prompt communication of serious adverse events and suspected 
unexpected serious adverse reactions (SUSARs) or serious safety-related protocol deviations 
between the sponsor, all study sites and investigators, patients and Competent Authorities and 
IECs according to the regulatory requirements. 
In the case of emerging safety issues, the Sponsor via Urgent Safety Measure (USM) 
dissemination shall inform Competent Authorities and IECs using the fastest means of 
communication followed by a written report as well as Investigators and participants (at any 
site) as soon as possible, and at least prior to any planned next dosing.  
USMs are a group of expedited actions that can be made in the exceptional circumstances of 
events occurred that can affect the participating subject’s safety and without prior approval of 
the relevant authorities, which aim is to protect the subjects against any immediate hazard. 
Details and procedures on USM management and dissemination are specified in the study 
Safety Management Plan. 
The Investigator must immediately inform the Sponsor in case of any relevant safety issue in 
the clinical trial which has the potential to expose subjects to an immediate hazard or any 
unexpected event that is likely to affect the benefit-risk ratio. Sponsor contacts DSMB for 
making a decision whether USM is necessary to protect participants against any immediate 


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hazard. If trial participants are at risk, the Sponsor redacts a written document for the urgent 
safety measure with the proposed corrective/preventative action to be disseminated to the trial 
Investigators immediately (i.e. within 24 hours of the decision) with acknowledgment of its 
receipt and implementation received from each Principal Investigator. 
 
 


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12 
ETHICS AND GENERAL STUDY ADMINISTRATION 
 Ethical aspects 
The investigator will ensure that this study is conducted in full conformity with either the 
principles of the "Declaration of Helsinki" (as amended in Tokyo, Venice, Hong Kong, South 
Africa and Edinburgh) or the laws and regulations of the country in which the study was 
conducted, whichever affords the greater protection to the individual. 
The protocol has been written and the study will be conducted in conformity with the "Guideline 
for Good Clinical Practice" and local regulations. 
 Independent Ethics Committees/Institutional Review Board 
This protocol and any accompanying material provided to the subject, such as subject 
information sheets or descriptions of the study used to obtain informed consent, will be 
submitted to an Institutional Review Board (IRB)/Independent Ethics Committee (IEC). 
Approval from the committee must be obtained before starting the study and should be 
documented in a letter specifying the date on which the committee met and granted the 
approval. 
Any modifications made to the protocol after receipt of the Independent Ethics Committee 
approval must also be submitted to the committee in accordance with local procedures and 
regulatory requirements. 
 Informed consent 
All patients will be informed of the aims of the study, the possible adverse events and the 
procedures and possible hazards to which they will be exposed. They will be informed as to the 
strict confidentiality of their patient data, but that their medical records may be reviewed for 
trial purposes by authorized individuals other than their treating physician. 
It will be emphasized that participation is voluntary and that the patient can refuse further 
participation in the protocol whenever he/she wants. This will not prejudice the patient’s 
subsequent care. 
It is the responsibility of the investigator or a person designated by the investigator (if 
acceptable by local regulations) to obtain written informed consent from each subject 
participating in this study before his/her registration in the trial. 
For patients unable to read, an impartial witness should be present during the entire informed 
consent discussion. After the subject and representative have orally consented to participation 
in the trial, the witness’ signature on the form will attest that the information in the consent 
form was accurately explained and understood. 
For minors (<18 years), parents or legal representatives must give permission by signing a 
specific informed consent form according to local laws. 
If new safety information results in significant changes in the risk/benefit assessment, the 
consent form is reviewed and approved, if necessary. All patients, including those already being 
treated, should be informed of the new information and give their consent to continue the study 


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after receiving a copy of the revised informed consent form. The Eligibility Screening Form 
ESF and CRF for this study contain a section for documenting informed patient consent and 
these must be completed appropriately. 
 Conditions for modifying the protocol 
Protocol modifications to ongoing studies must be made only after consultation between an 
appropriate representative of the sponsor and the investigator. Protocol modifications must be 
discussed by a representative of the sponsor and the principal investigator. Protocol 
modifications must be prepared by a representative of the sponsor and initially reviewed and 
approved by the Sponsor. 
All protocol modifications must be submitted to the appropriate regulatory authorities, 
Independent Ethics Committee and/or Ministry of Health for information and approval in 
accordance with local requirements. Approval must be obtained before any changes can be 
implemented except for changes necessary to eliminate an immediate hazard to trial subjects or 
when the change(s) involve only logistical or administrative aspect of the trial. 
 Conditions for terminating the study 
Both the sponsor and the investigator reserve the right to terminate the study at any time. Should 
this be necessary both parties will arrange the procedures on an individual study basis after 
review and consultation. In terminating the study, MolMed and the investigators will assure 
that adequate consideration is given to the protection of the patient’s interest. 
 Study documentation: CRF and record keeping 
The investigator must maintain adequate and accurate records to enable the conduct of the study 
to be fully documented and the study data to be subsequently verified. These documents should 
be classified into 2 different categories: 
 Investigator’s study file 
 Subject clinical source documents 
The investigators’ study file will contain the protocol/amendments, CRF (Case Report Form) 
and query forms, IRB/IEC approvals with correspondence, sample of informed consent, 
drug/product records, staff CVs and authorization forms and other appropriate 
documents/correspondence. 
Subject clinical source documents include patient hospital/clinic records, physician’s and nurse 
notes, appointment book, original laboratory reports, ECG, EEG, x-ray, pathology and special 
assessment reports, signed informed consent forms, consultant letters and subject screening and 
enrolments logs. The investigator must keep these two categories of documents on file until at 
least 2 years after the last approval of a marketing application or at least 15 years after 
completion or discontinuation of the study. After that period, the documents may be destroyed, 
subject to local regulations. 
All traceability records should be kept for a minimum of 30 years after the end of study by each 
party: lymphocyte apheresis establishments/procurement, manufacturer, Sponsor and 


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investigator/clinical site, as outlined in the annex of “Detailed guidelines on good clinical 
practice specific to advanced therapy medicinal products” and in Regulation 1394/2007. 
Should the investigator not be able to guarantee this archiving requirement at the investigational 
site for any or all the documents special arrangements must be made between the investigator 
and MolMed to store these in a sealed container outside of the site so that they can be returned 
sealed to the investigator in case of regulatory audit. Where source documents are required for 
the continued care of the patient, appropriate copies should be made for storing outside of the 
site. 
 Source documents and background data 
The investigator shall supply the sponsor with any required background data from the study 
documentation or clinical records. In case of special problems and/or governmental queries or 
request for audit inspections, it is also necessary to have access to the complete study records 
provided that patient confidentiality is protected. 
 Audits and inspections 
The investigator should understand that source documents for this trial should be made 
available to appropriately qualified personnel from the MolMed Quality Assurance Unit or its 
designees or to health authorities after appropriate notification. 
 Case Report Forms 
For each patient enrolled, an electronic case report form (eCRF) must be completed by the 
principal investigator or one of his/her authorized staff members within 5 days from the clinical 
visit.  
If a patient withdraws from the study the reason must be noted on the eCRF. 
If a patient is withdrawn from the study because of a treatment limiting adverse event, thorough 
efforts should be made to clearly document the outcome. 
The investigator should ensure the accuracy, completeness and timelines of the data reported 
to the sponsor in the eCRF and in all required reports.  
eCRF instructions will be provided in a detailed manual. 
 Monitoring the study 
It is understood that the MolMed monitor or designee will contact and visit the investigator 
regularly and will be allowed on request to inspect the various records of the trial provided that 
patient confidentiality is maintained in accord with local requirements. 
It will be the monitor’s responsibility to inspect the CRFs at regular intervals throughout the 
study to verify the adherence to the protocol and the completeness, consistency and accuracy 
of the data being entered on them. The monitor should have access to laboratory test reports 
and other patient records needed to verify the entries on the CRF. 


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The investigator agrees to cooperate with the monitor to ensure that any problems detected 
during these monitoring visits are resolved. 
The end of study in a center corresponds to the last patient’s last visit. 
The end of the completion of the trial in all countries corresponds to the date of the end of study 
at the last center.  
 Confidentiality of trial documents and subject records 
The investigator must assure that subjects’ anonymity will be maintained and that their 
identities are protected from unauthorized parties. On CRFs or other documents submitted to 
the sponsor, subjects should not be identified by their names but by an identification code. The 
investigator should keep a subject enrolment log showing code names. The investigator should 
maintain documents not for submission to MolMed (e.g. written informed consent) in strict 
confidence. 
 Publication of data and protection of trade secrets 
The results of this study may be published or presented at scientific meetings. In this case the 
investigator agrees to discuss all manuscripts or abstracts with MolMed prior to submission. 
This allows the sponsor to protect proprietary information and to provide comments based on 
information from other studies that may not be yet available to the investigator. EURE CAR T 
requirements for publication and dissemination will also be considered. 
In accord with standard editorial and ethical practice, MolMed will generally support 
publication of multicentric trials only in their entirety and not as individual centre data. 
Authorship will be determined by agreement. 
 
 


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13 
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 Pule MA, Straathof KC, Dotti G et al. A chimeric T cell antigen receptor that augments 
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expression but has no consequence on the biology and function of transplanted T cells. 
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migration, and survival offers a means for therapeutic attack. J Immunol. 2013; 
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Appendix 1: BOIN design 
 
We performed a simulation study in the specific context we are evaluating to 
1) define the most suitable sample size for the trial. 
2) assess the operating characteristics at the chosen sample size. 
We considered a BOIN design with the same parameters of the current study: 3 doses, cohorts 
of 3 patients, a target toxicity at 30%, a tolerance toxicity interval of 20%-40%, identifying an 
optimal interval for decision of 24.7%-34.8% and a value for overdose control of 95%.  
We simulated 2000 potential trial configurations, using seven different scenarios of toxicity 
assigned to the doses as follows: 
 
Scenario 
D1
D2
D3
1 ALL 
TOX 
0.45 
0.55 
0.60 
2 D1 
0.30 
0.45 
0.55 
3 D1/D2 
0.20 
0.40 
0.50 
4 D2 
0.15 
0.30 
0.50 
5 D2/D3 
0.15 
0.25 
0.35 
6 D3 
0.07 
0.15 
0.30 
7 NO TOX
0.01 
0.10 
0.15 
 
Specifically, i) the first and last scenarios reflect the extreme cases where all or no doses are 
toxic, ii) scenarios 2, 4 and 6 correspond to MTD exactly at D1, D2 and D3, and iii) in scenarios 
3 and 5 the MTD is included between two consecutive doses (i.e. D1-D2 and D2-D3) and thus 
not directly assigned to a specific dose. 
Evaluation of sample size 
To compare the performances of the BOIN design at different sample sizes (n=12, 15, 18) we 
considered the accuracy in MTD selection. The results in terms of percentage of MTD selection 
at each dose level are shown in Figure 1. 
 
 


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FIGURE 1: PERCENTAGE OF MTD SELECTION AT EACH DOSE LEVEL AND EARLY TERMINATION 
(WITHOUT SELECTING MTD) IN 7 SCENARIOS OF TOXICITY WITH N=12,15,18 
 
As regards safety, trials are terminated when high levels of toxicity are seen (scenario 1) and 
the percentage of early termination increase, increasing sample size. Conversely, in the absence 
of toxicity (scenario 7), larger samples increase the chance of identifying the last dose as MTD. 
In general, results are similar for n=15 and 18, while the worst performances are consistently 
seen with n=12. In light of these results, a sample size of 15 patients was defined for the EURE-
CART-1 study.  
Operating characteristics 
The operating characteristics of the BOIN design for the EURE CAR T-1 trial with n=15 are 
shown in the table below.   
 
 
Dose level     
% early stopping     
Number of pts 
1
2
3
1 
True DLT rate 
0.5 
0.6 
0.6 
75.8 
7.7 
 
Selection % 
19.3 
4.5 
0.5 
 
# pts treated 
6.0 
1.5 
0.2 
2 
True DLT rate 
0.3 
0.5 
0.6 
39.7 
11.4 
 
Selection % 
39.7 
17.4 
3.3 
 
# pts treated 
7.1 
3.6 
0.7 
3 
True DLT rate 
0.2 
0.4 
0.5 
17.2 
13.3 
 
Selection % 
45.9 
28.3 
8.7 


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Dose level     
% early stopping     
Number of pts 
1
2
3
 
# pts treated 
6.7 
5.2 
1.3 
4 
True DLT rate 
0.2 
0.3 
0.5 
8.9 
14.1 
 
Selection % 
29.4 
47.1 
14.7 
 
# pts treated 
5.7 
6.0 
2.4 
5 
True DLT rate 
0.15 
0.25 
0.5 
8.9 
14.1 
 
Selection % 
18.9 
38.3 
34.0 
 
# pts treated 
5.3 
5.5 
3.3 
6 
True DLT rate 
0.1 
0.2 
0.3 
1.9 
14.8 
 
Selection % 
4.9 
34.9 
58.4 
 
# pts treated 
3.9 
5.2 
5.7 
7 
True DLT rate 
0.0 
0.1 
0.2 
0 
15.0 
 
Selection % 
0.9 
9.7 
89.4 
 
# pts treated 
3.2 
4.3 
7.5 
 
The operating characteristics show that the BOIN design selects the true MTD with high 
probabilities and allocates more patients to the dose levels with the DLT rate closest to the 
target of 0.3.  
In Figure 2A we have also reported the most common potential trial configurations (≥1%) in 
scenarios 4, from the most to the less probable, with the occurrence (n, %) reported in each 
pattern. In each scatter-plot the dose escalation process and the MTD selection following the 
BOIN design are shown. The x and y axes represent patients and doses, respectively, while the 
individual DLT response to treatment are reported as: no toxicity=filled circle and 
toxicity=cross. The horizontal red line corresponds to the selected MTD. 
FIGURE 2: MOST COMMON TOXICITY PATTERNS SEEN IN THE SIMULATION OF SCENARIO 4. 
NUMBERS OUTSIDE THE PLOTS INDICATE THE SIMULATION ID 
 


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Appendix 2: International Staging System (ISS) and revised-ISS (R-
ISS) for multiple myeloma 
 
 
 
 
Palumbo et al. 2015 
 
 


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Appendix 3: European LeukemiaNet (ELN) Response Criteria for 
Acute Myeloid Leukemia 
 
Category 
Definition 
Comment 
Response 
CR without 
minimal 
residual disease 
(CRMRD−) 
If studied pretreatment, CR with negativity for a 
genetic marker by RT-qPCR, or CR with 
negativity by MFC 
Sensitivities vary by marker 
tested and by method used. 
Complete 
remission (CR) 
Bone marrow blasts <5%; absence of circulating 
blasts and blasts with Auer rods; absence of 
extramedullary disease; ANC ≥1.0 × 109/L 
(1000/µL); platelet count ≥100 × 109/L 
(100 000/µL) 
MRD+ or unknown 
CR with 
incomplete 
hematologic 
recovery (CRi) 
All CR criteria except for residual neutropenia 
(<1.0 × 109/L [1000/µL]) or thrombocytopenia 
(<100 × 109/L [100 000/µL]) 
 
Morphologic 
leukemia-free 
state (MLFS) 
Bone marrow blasts <5%; absence of blasts 
with Auer rods; absence of extramedullary 
disease; no hematologic recovery required 
Marrow should not merely be 
“aplastic”; at least 200 cells 
should be enumerated or 
cellularity should be at least 
10% 
Partial 
remission (PR) 
All hematologic criteria of CR; decrease of 
bone marrow blast percentage to 5% to 25%; 
and decrease of pretreatment bone marrow blast 
percentage by at least 50% 
Especially important in the 
context of phase 1-2 clinical 
trials 
Treatment failure 
Primary 
refractory 
disease 
No CR or CRi after 2 courses of intensive 
induction treatment; excluding patients with 
death in aplasia or death due to indeterminate 
cause 
 
Death in aplasia 
Deaths occurring ≥7 d following completion of 
initial treatment while cytopenic; with an 
aplastic or hypoplastic bone marrow obtained 
within 7 d of death, without evidence of 
persistent leukemia 
 
Death from 
indeterminate 
cause 
Deaths occurring before completion of therapy, 
or <7 d following its completion; or deaths 
occurring ≥7 d following completion of initial 
therapy with no blasts in the blood, but no bone 
marrow examination available 
 
Response criteria for clinical trials only 
Stable disease 
Absence of CRMRD−, CR, CRi, PR, MLFS; and 
criteria for PD not met 
Period of stable disease 
should last at least 3 months 
Progressive 
disease (PD)*,† 
Evidence for an increase in bone marrow blast 
percentage and/or increase of absolute blast 
counts in the blood: 
Category mainly applies for 
older patient given low-
intensity or single-agent 
“targeted therapies” 


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Category 
Definition 
Comment 
• >50% increase in marrow blasts over baseline 
(a minimum 15% point increase is required in 
cases with <30% blasts at baseline; or persistent 
marrow blast percentage of >70% over at least 3 
mo.; without at least a 100% improvement in 
ANC to an absolute level (>0.5 × 109/L 
[500/µL], and/or platelet count to >50 × 109/L 
[50 000/µL] nontransfused); or 
In general, at least 2 cycles of 
a novel agent should be 
administered 
• >50% increase in peripheral blasts (WBC × % 
blasts) to >25 × 109/L (>25 000/μL) (in the 
absence of differentiation syndrome)†; or 
Some protocols may require 
blast increase in 2 
consecutive marrow 
assessments at least 4 wk 
apart; the date of progression 
should then be defined as of 
the first observation date 
• New extramedullary disease 
Some protocols may allow 
transient addition of 
hydroxyurea to lower blast 
counts 
“Progressive disease” is 
usually accompanied by a 
decline in ANC and platelets 
and increased transfusion 
requirement and decline in 
performance status or 
increase in symptoms 
Relapse 
Hematologic 
relapse (after 
CRMRD−, CR, 
CRi) 
Bone marrow blasts ≥5%; or reappearance of 
blasts in the blood; or development of 
extramedullary disease 
 
If studied pretreatment, reoccurrence of MRD 
as assessed by RT-qPCR or by MFC 
Test applied, sensitivity of the 
assay, and cutoff values used 
must be reported. 
From Dohner et al., Blood 2017; 129(4): 424-447 
ANC, absolute neutrophil count; IDH, isocitrate dehydrogenase; MLFS, morphologic leukemia-free state; WBC, white blood cell. 
*The authors acknowledge that this new provisional category is arbitrarily defined; the category aims at harmonizing the various 
definitions used in different clinical trials. 
†Certain targeted therapies, for example those inhibiting mutant IDH proteins, may cause a differentiation syndrome, that is, a 
transient increase in the percentage of bone marrow blasts and an absolute increase in blood blasts; in the setting of therapy with 
such compounds, an increase in blasts may not necessarily indicate PD. 
 
 
 


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Appendix 4: Modified International Myeloma Working Group 
(IMWG) Uniform Response Criteria for Multiple Myeloma 
Response 
IMWG criteria 
Stringent complete 
response (sCR) 
CR as defined below plus normal FLC ratio and absence of clonal cells in bone 
marrow1 by immunohistochemistry or immunofluorescence.2 
Complete response 
(CR) 
Negative immunofixation on serum and urine and disappearance of any soft 
tissue plasmacytomas and < 5% plasma cells in bone marrow.1 
Very good partial 
response (VGPR) 
Serum and urine M-protein detectable by immunofixation but not on 
electrophoresis or > 90% reduction in serum M-protein plus urine M-protein 
level < 100 mg/24 h 
Partial response 
(PR) 
> 50% reduction of serum M-protein and reduction in 24 hours urinary M-
protein by >90% or to < 200 mg/24 h. 
If serum and urine M-protein are unmeasurable, 3 a > 50% decrease in the 
difference between involved and uninvolved FLC levels is required in place of 
the M-protein criteria. 
If serum and urine M-protein are not measurable, and serum free light assay is 
also not measurable, > 50% reduction in plasma cells is required in place of M 
protein, provided baseline bone marrow plasma cell percentage was > 30%. 
In addition to the above listed criteria, if present at baseline, a > 50% reduction 
in the size of soft tissue plasmacytomas is also required. 
Minor (Minimal) 
Response (MR) 
25-49% reduction of serum M-protein and reduction in 24-hour urine M-protein 
by 50-89%, which still exceeds 200 mg per 24 hours. In addition, if present at 
baseline, 25-49% reduction in the size of soft tissue plasmacytomas is also 
required. No increase in the size or number of lytic bone lesions (development 
of compression fracture does not exclude response). 
No change/Stable 
disease (SD) 
Not meeting criteria for CR, VGPR, PR, or progressive disease. 
Progressive 
disease (PD)3 
Any of the following: 
 
Increase of > 25% from lowest response value in any one or more of the 
following: 
o 
Serum M-component and/or (the absolute increase must be > 0.5 g/dL)4 
o 
Urine M-component and/or (the absolute increase must be > 200 mg/24 
h) 
o 
Only in patients without measurable serum and urine M-protein levels; 
the difference between involved and uninvolved FLC levels. The 
absolute increase must be > 10 mg/dL 
o 
Bone marrow plasma cell percentage; the absolute percentage must be 
> 10%5 
 
Definite development of new bone lesions or soft tissue plasmacytomas or 
definite increase in the size of existing bone lesions or soft tissue 
plasmacytomas (≥50% increase from nadir in SPD7 of >1 lesion, or ≥50% 
increase in the longest diameter of a previous lesion >1 cm in the short axis).


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Response 
IMWG criteria 
 
Development of hypercalcemia (corrected serum calcium > 11.5 mg/dL or 
2.87 mmol/L) that can be attributed solely to the plasma cell proliferative 
disorder 
Relapse 
Clinical relapse requires one or more of: 
Direct indicators of increasing disease and/or end organ dysfunction (CRAB 
features).4 It is not used in calculation of time to progression or progression-
free survival but is listed here as something that can be reported optionally or 
for use in clinical practice 
 
Development of new soft tissue plasmacytomas or bone lesions 
 
Definite increase in the size of existing plasmacytomas or bone lesions. A 
definite increase is defined as a 50% (and at least 1 cm) increase as 
measured serially by the sum of the products of the cross-diameters of the 
measurable lesion 
 
Hypercalcemia (> 11.5 mg/dL) [2.87 mmol/L] 
 
Decrease in hemoglobin of > 2 g/dL [1.24 mmol/L] 
 
Rise in serum creatinine by 2 mg/dL or more [177 mol/L or more] 
Relapse from CR3 
(To be used only if 
the 
endpoint 
studied is DFS)6  
Any one or more of the following: 
 
Reappearance of serum or urine M-protein by immunofixation or 
electrophoresis 
 
Development of > 5% plasma cells in the bone marrow5 
 
Appearance of any other sign of progression (i.e., new plasmacytoma, lytic 
bone lesion, or hypercalcemia) 
Note: A clarification to IMWG criteria for coding CR and VGPR in patients in whom the only 
measurable disease is by serum FLC levels: CR in such patients is defined as a normal FLC ratio of 
0.26–1.65 in addition to CR criteria listed above. VGPR in such patients is defined as a >90% decrease 
in the difference between involved and uninvolved free light chain (FLC) levels. 
1 Confirmation with repeat bone marrow biopsy not needed. 
2 Presence/absence of clonal cells is based upon the kappa/lambda ratio. An abnormal kappa/lambda 
ratio by immunohistochemistry and/or immunofluorescence requires a minimum of 100 plasma cells for 
analysis. An abnormal ratio reflecting presence of an abnormal clone is kappa/lambda of > 4:1 or < 1:2. 
3 All relapse categories require two consecutive assessments made at any time before classification as 
relapse or disease progression and/or the institution of any new therapy. In the IMWG criteria, CR 
patients must also meet the criteria for progressive disease shown here to be classified as progressive 
disease for the purposes of calculating time to progression and progression-free survival. The definitions 
of relapse, clinical relapse and relapse from CR are not to be used in calculation of time to progression 
or progression-free survival. 
4 For progressive disease, serum M-component increases of >1 gm/dL are sufficient to define relapse if 
starting M-component is >5 g/dL. 
5 Relapse from CR has the 5% cut-off versus 10% for other categories of relapse. 
6 For purposes of calculating time to progression and progression-free survival, CR patients should also 
be evaluated using criteria listed above for progressive disease. 
7 SPD = sum of the products of the maximal perpendicular diameters of measured lesions 
 
 


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Appendix 5: WHO classification of myeloid leukemia 
 
AML with certain genetic abnormalities 
 
AML with a translocation between chromosomes 8 and 21 
 
AML with a translocation or inversion in chromosome 16 
 
APL with the PML-RAPA fusion gene 
 
AML with a translocation between chromosomes 9 and 11 
 
AML with a translocation between chromosomes 6 and 9 
 
AML with a translocation or inversion in chromosome 3 
 
AML (megakaryoblastic) with a translocation between chromosomes 1 and 22 
 
AML with the BCR-ABL1 (BCR-ABL) fusion gene* 
 
AML with mutated NPM1 gene 
 
AML with biallelic mutations of the CEBPA gene (that is, mutations in both copies of 
the gene) 
 
AML with mutated RUNX1 gene 
*This is still a "provisional entity," meaning it's not yet clear if there's enough evidence that it's 
a unique group 
 
AML with myelodysplasia-related changes 
 
AML related to previous chemotherapy or radiation 
 
AML not otherwise specified (This includes cases of AML that don’t fall into one of the above 
groups and is similar to the FAB classification.) 
 
AML with minimal differentiation (M0) 
 
AML without maturation (M1) 
 
AML with maturation (M2) 
 
Acute myelomonocytic leukemia (M4) 
 
Acute monocytic leukemia (M5) 
 
Acute erythroid leukemia (M6) 
 
Acute megakaryoblastic leukemia (M7) 
 
Acute basophilic leukemia 
 
Acute panmyelosis with fibrosis 
 
Myeloid sarcoma (also known as granulocytic sarcoma or chloroma) 
 
Myeloid proliferations related to Down syndrome 
 
Undifferentiated and biphenotypic acute leukemias (leukemias that have both lymphocytic 
and myeloid features). Sometimes called ALL with myeloid markers, AML with lymphoid 
markers, or mixed phenotype acute leukemias. 
 
 
 


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Appendix 6: ECOG performance status scale 
 
Grade 
Performance scale 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g. light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work 
activities; up and about more than 50% of waking hours 
3 
Capable of only limited self-care; confined to bed or chair more than 50% of 
waking hours 
4 
Completely disabled; cannot carry on any self-care; totally confined to bed or 
chair 
5 
Dead  
 
* As published in Am. J. Clin. Oncol.: Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., 
McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. 
Am J Clin Oncol 5:649-655, 1982. 
 
 
 


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Appendix 7: Grading of CAR-T-cell-related encephalopathy syndrome 
(CRES) 
 
Symptom or 
sign 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Neurological 
assessment 
score 
(CARTOX-
10*) 
7-9 (mild 
impairment) 
3-6 (moderate 
impairment) 
0-2 (severe 
impairment 
Patient in critical 
condition, and/or 
obtunded and cannot 
perform assessment 
of tasks 
Raised 
intracranial 
pressure 
NA 
NA 
Stage 1–2 
papilloedema‡, or 
CSF 
opening pressure <20 
mmHg 
Stage 3–5 
papilloedema‡, or 
CSF opening 
pressure ≥20 mmHg, 
or cerebral oedema 
Seizures or 
motor weakness 
NA 
NA 
Partial seizure, or 
non‑convulsive 
seizures on EEG with 
response to 
benzodiazepine 
Generalized seizures, 
or convulsive or 
non‑convulsive status 
epilepticus, or new 
motor weakness 
CAR, chimeric antigen receptor; CARTOX‑10, CAR‑T‑cell‑therapy associated toxicity 10‑point neurological 
assessment CSF, cerebrospinal fluid; EEG, electroencephalogram; NA, not applicable. 
*In the CARTOX‑10, one point is assigned for each of the following tasks that is performed correctly (normal 
cognitive function is defined by an overall score of 10): orientation to year, month, city, hospital, and 
President/Prime Minister of country of residence (total of 5 points); name three objects — for example, point to 
clock, pen, button (maximum of 3 points); write a standard sentence, for example, ‘our national bird is the bald 
eagle’ (1 point); count backwards from 100 in tens (1 point). 
‡Papilloedema grading is performed according to the modified Frisén scale98. 
 
 
 


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Appendix 8: Common terminology criteria for Adverse Events 
 
In the present study adverse events and/or adverse drug reactions will be recorded according to 
the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. 
At the time this protocol was issued, the full CTC document was available on the NCI web site, 
at the following address: 
http://ctep.cancer.gov/reporting/ctc.html 


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Appendix 9: 2017 European LeukemiaNet (ELN) recommendations 
for diagnosis and management of AML 
 
AML and related precursor neoplasms, and acute leukemias of ambiguous lineage (WHO 
2016) 
 
For a diagnosis of AML, a marrow blast count of $20% is required, except for AML with the recurrent genetic abnormalities t(15;17), 
t(8;21), inv(16), or t(16;16). Adapted from Arber et al.3 
MPAL, mixed phenotype acute leukemia; NK, natural killer. 
*Other recurring translocations involving RARA should be reported accordingly: for example, AML with t(11;17)(q23;q12); ZBTB16-
RARA; AML with t(11;17)(q13;q12); NUMA1-RARA; AML with t(5;17)(q35;q12); NPM1-RARA; or AML with STAT5B-RARA (the 
latter having a normal chromosome 17 on conventional cytogenetic analysis). 
†Other translocations involving KMT2A (MLL) should be reported accordingly: for example, AML with t(6;11)(q27;q23.3); MLLT4-
KMT2A; AML with t(11;19)(q23.3;p13.3); KMT2A-MLLT1; AML with t(11;19)(q23.3;p13.1); KMT2A-ELL; AML with 
t(10;11)(p12;q23.3); MLLT10-KMT2A. 
‡Rare leukemia most commonly occurring in infants. 
§Diagnosis is made irrespective of the presence or absence of multilineage dysplasia. 
||At least 20% ($20%) blood or marrow blasts AND any of the following: previous history of MDS or MDS/MPN; myelodysplasia-
related cytogenetic abnormality (see list below); multilineage dysplasia; AND absence of both prior cytotoxic therapy for unrelated 
disease and aforementioned recurring genetic abnormalities. Cytogenetic abnormalities sufficient to diagnose AML with 
myelodysplasia-related changes are: Complex karyotype (defined as 3 or more chromosomal abnormalities in the absence of 1 of the 
WHO-designated recurring translocations or inversions, that is, t(8;21), inv(16) or t(16;16), t(9;11), t(v;11)(v;q23.3), t(6;9), inv(3) or 
t(3;3); AML with BCR- ABL1); Unbalanced abnormalities: 27 or del(7q); 25 or del(5q); i(17q) or t(17p); 213 or del(13q); del(11q); 
del(12p) or t(12p); idic(X)(q13); Balanced abnormalities: t(11; 16)(q23.3;p13.3); 
t(3;21)(q26.2;q22.1); 
t(1;3)(p36.3;q21.2); 
t(2;11)(p21;q23.3);  t(5;12)(q32;p13.2); t(5;7)(q32;q11.2); t(5;17)(q32;p13.2); t(5;10)(q32;q21.2); t(3;5) (q25.3;q35.1). 
{Cases should be classified with the related genetic abnormality given in the diagnosis. 
#The former subgroup of acute erythroid leukemia, erythroid/myeloid type ($50% bone marrow erythroid precursors and $20% 
myeloblasts among nonerythroid cells) was removed; myeloblasts are now always counted as percentage of total marrow cells. The 
remaining subcategory AML, NOS, pure erythroid leukemia requires the presence of .80% immature erythroid precursors with $30% 
proerythroblasts. 
**BCR-ABL11 leukemia may present as MPAL; treatment should include a tyrosine kinase inhibitor. 
 
 


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2017 ELN risk stratification by genetics 
 
Frequencies, response rates, and outcome measures should be reported by risk category, and, if sufficient numbers are available, by specific 
genetic lesions indicated. 
*Prognostic impact of a marker is treatment-dependent and may change with new therapies. 
†Low, low allelic ratio (,0.5); high, high allelic ratio ($0.5); semiquantitative assessment of FLT3-ITD allelic ratio (using DNA 
fragment analysis) is determined as ratio of the area under the curve “FLT3-ITD” divided by area under the curve “FLT3- wild type”; recent 
studies indicate that AML with NPM1 mutation and FLT3-ITD low allelic ratio may also have a more favorable prognosis and patients 
should not routinely be assigned to allogeneic HCT.57-59,77 
‡The presence of t(9;11)(p21.3;q23.3) takes precedence over rare, concurrent adverse-risk gene mutations. 
§Three or more unrelated chromosome abnormalities in the absence of 1 of the WHO-designated recurring translocations or inversions, 
that is, t(8;21), inv(16) or t(16;16), t(9;11), t(v;11)(v;q23.3), t(6;9), inv(3) or t(3;3); AML with BCR-ABL1. 
||Defined by the presence of 1 single monosomy (excluding loss of X or Y) in association with at least 1 additional monosomy or 
structural chromosome abnormality (excluding core-binding factor AML).116 
{These markers should not be used as an adverse prognostic marker if they co- occur with favorable-risk AML subtypes. 
#TP53 mutations are significantly associated with AML with complex and monosomal karyotype.37,66-69 
 
 


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Response criteria in AML 
 
ANC, absolute neutrophil count; IDH, isocitrate dehydrogenase; MLFS, morphologic leukemia-free state; WBC, white blood cell. 
*The authors acknowledge that this new provisional category is arbitrarily defined; the category aims at harmonizing the various 
definitions used in different clinical trials. 
†Certain targeted therapies, for example, those inhibiting mutant IDH proteins, may cause a differentiation syndrome, that is, a transient 
increase in the percentage of bone marrow blasts and an absolute increase in blood blasts; in the setting of therapy with such compounds, an 
increase in blasts may not necessarily indicate PD. 
 


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Selected conventional care regimens for patient with AML 
 
Patients should go on clinical trials if possible. 
EMA, European Medicines Agency; FLAG-AMSA, FLAG 1 amsacrine; FLAG-MITO, FLAG 1 mitoxantrone; q, every; SC, 
subcutaneously. 
*Regimens containing higher doses of cytarabine are generally considered as the best option for patients not responding to a first cycle 
of “713” (see common salvage regimens). 
†Older patients (in general .65 y) and patients with adverse genetics are less likely to respond to conventional induction therapy and may 
receive hypomethylating agents, or, preferably, investigational therapy. 
‡Patients, at least those aged 18 to 60 y, with newly diagnosed AML and activating FLT3 mutations may be considered to receive 
additional therapy with midostaurin (administered after the chemotherapy).61 
§Results from assessment of MRD should be taken into account for selecting the appropriate consolidation therapy. 
||For discussion of patients not considered candidates for intensive chemotherapy see first 2 paragraphs of “Current therapy.” 
{Approved by FDA and EMA for adult patients who are not eligible for HCT with AML with 20% to 30% blasts and multilineage 
dysplasia; in addition, approved by EMA for patients who are not eligible for allogeneic HCT with AML with .30% marrow blasts. 
#Approved by EMA (not by FDA) for patients with newly diagnosed de novo or secondary AML, who are not candidates for standard 
induction chemotherapy. 
**In some countries used in a dosage of 20 mg/m2 SC once daily. 
††Evidence from pharmacologic studies and clinical trials in first-line treatment indicate that doses higher than 1500 mg/m2  are above 
the plateau of the maximal therapeutic effect;147 single-agent IDAC should not be used in patients relapsing within 6 mo following 
consolidation with higher doses of cytarabine. 
‡‡Idarubicin may be replaced by mitoxantrone 10 mg/m2, IV, days 2 to 4 (FLAG-MITO); or by amsacrine 100 mg/m2, days 2 to 4 
(FLAG-AMSA). 
 


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Appendix 10: Grant members 
 
This Agreement (‘the Agreement’) is between the following parties: 
on the one part, the European Union (‘the EU’), represented by the European Commission 
('the Commission')1, represented for the purposes of signature of this Agreement by the Head 
of Unit, DIRECTORATE GENERAL FOR RESEARCH & INNOVATION, Health, 
Administration and finance, Mila BAS SANCHEZ, and on the other part, 
1. ‘the coordinator’: 
MOLECULAR MEDICINE SPA (MLM) SPA, 1506630, established in VIA OLGETTINA 
58, MILANO 20132, Italy, VAT number IT11887610159, represented for the purposes of 
signing the Agreement by CEO, by Riccardo PALMISANO and the following other 
beneficiaries, if they sign their ‘Accession Form’ (see Annex 3 and Article 56): 
2. OSPEDALE SAN RAFFAELE SRL (OSR) SRL, 1972938, established in VIA 
OLGETTINA 60, MILANO 20132, Italy, VAT number IT07636600962, 
3. UNIVERSITAETSKLINIKUM WUERZBURG - KLINIKUM DER BAYERISCHEN 
JULIUS-MAXIMILIANS-UNIVERSITAT (UKW), not applicable, established in 
JOSEFSCHNEIDER- STRASSE 2, WURZBURG 97080, Germany, VAT number 
DE250013752, 
4. OSPEDALE PEDIATRICO BAMBINO GESU (OPBG), -, established in PIAZZA SANT 
ONOFRIO 4, ROMA 00165, Italy, VAT number VAT exemption, 
5. FUNDACIO PRIVADA INSTITUT DE RECERCA DE L'HOSPITAL DE LA SANTA 
CREU I SANT PAU (PAU) ES3, 708, established in CALLE SANT ANTONI M CLARET 
167, BARCELONA 08025, Spain, VAT number ESG60136934, 
6. FAKULTNI NEMOCNICE S POLIKLINIKOU OSTRAVA FOUNDATION (UHO), 
00843989, established in 17 LISTOPADU 1790, OSTRAVA PORUBA 708 52, Czech 
Republic, VAT number CZ00843989, 
7. ISTITUTO SUPERIORE DI SANITA (ISS), 80211730587, established in Viale Regina 
Elena 299, ROMA 00161, Italy, VAT number IT03657731000, 
8. ACROMION GMBH (ACRO) GMBH, HRB45379, established in EUROPAALLEE 27-
29, FRECHEN 50226, Germany, VAT number DE224978890, 
9. ARTTIC (ART) SAS, 344112396, established in RUE DU DESSOUS DES BERGES 58A, 
PARIS 75013, France, VAT number FR53344112396, 
 
 


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Appendix 11: Amendment history 
 
Protocol Version 
Protocol Approval Date 
Rationale and Substantial Changes 
A 
27 August 2019 
First release 
B 
22 July 2019 
Version released for the German clinical center, but not 
implemented as not approved by the Competent Authority 
of Germany (PEI). 
C 
8 January 2020 
Version 
released 
to 
implement 
mainly 
more 
details/clarifications on study procedures, as follows: 
 
Section 3.1: it has better clarified that the first 3 
patients of all cohorts will be treated according to 
the staggered approach 
 
Section 4.2 inclusion criteria 3: measurable criteria 
for MM disease have been defined 
 
Section 4.2 inclusion criteria 5: it has been defined 
the threshold value of  ≥ 20%  for CD44v6 
positivity on tumor cells  
 
Section 4.4: no treatment with steroids at least 3 
days before lymphocyte apheresis has been added 
 
Section 5.5.1: Toxic Epidermal Necrolysis (TEN) 
has been included as DLT. All sections on DLTs 
have been updated. 
 
Section 5.6: it has been defined the amount of 
CD3+ cells required for the lymphocyte apheresis 
collection 
 
Section 5.7: it has been better defined the 
possibility to administer salvage chemotherapy  
 
Section 
5.8: 
it 
has 
been 
included 
the 
communication to Sponsor of any delay ≥ 1 week 
for the lymphodepleting chemotherapy. Timeline 
for execution of rapid influenza diagnostic test has 
been update. 
 
Section 5.9: it has been specified the delay for up 
10 days for the MLM-CAR44.1 T-cell infusion 
and the recommendation of  GCV/valGCV wash-
out  at least 48 h prior to infusion 
 
Section 5.10: it has been included the immune 
phenotype analysis of MLM-CAR44.1 T-cells in 
case of GCV/valGCV administration as rescue 
medication (day 0, 4 and at the end of admin.) 
 
Section 5.12.1.1: it has been better defined the 
criteria for interrupting/discontinuing the study 
 
Section 6.1.1.1/6.1.1.1: it has been included the 
possibility of patient re-screening in exceptional 
cases 
 
Section 6.1.1.2/6.2.1.2 and 8: it has been better 
detailed the CD44v6 assessment for AML patients 
and MM patients 
 
Section 6.2: assessment of CD44v6 expression on 
tumor cells in PB and BM has been included also 
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 
Section 6.2: R-ISS assessment has been also 
included. (appendix 2 has been updated) 
 
Section 6.5: it has been better clarified the RCR 
collection/analysis during the Long-Term Follow-
up 
 
Section 8.4.2: it has been added the evaluation of 
urine FLC  
 
Section 8.4.3: MM markers have been added 
 
Section 8.4.8: more details on FISH assessments 
have been added  
 
Section 8.6 Chemistry: IgA, IgG and IgM have 
been included 
 
Section 9.1: it has been included the definition of 
“evaluable” and “not evaluable” patient  
 
Chapter 10: DSMB information has been updated 
according to the Data Safety Monitoring Board 
Charter 
 
Section 11.6.1.4: recommendations in case of skin 
rash 
have 
been 
added. 
A 
clear 
and 
a 
comprehensive 
description 
of 
symptoms/diagnostic markers for TEN and SJS, 
including appropriate treatment management, has 
been detailed. 
 
Section 11.6.2: procedures for ensuring the prompt 
communication of significant safety matters 
(Urgent Safety Measures) between the sponsor, all 
study sites and investigators and trial subjects have 
been added. 
 
Section 12.9: timelines for eCRF completion have 
been added. 
 
Appendix 9: guidance for definition of high risk 
leukemia in adults has been added 
In addition, this version includes also the changes required 
by the State Institute for Drug Control (Státní ústav pro 
kontrolu léčiv) of Czech Republic (CZ), previously 
implemented in a local protocol version (IPR/33.A-LA-
001). The protocol and corresponding sections of the 
synopsis have been modified accordingly as follows: 
 
Pediatric patients with AML expected in phase II, 
will not be enrolled in CZ 
 
patients enrolled in Phase I must be enrolled 
between the ages of 18 and 63 years. Following a 
substantial amendment and transition to Phase IIa, 
all adult patients may be enrolled without further 
age restriction.