CD19 CAR-T Expressing IL7 and CCL19 Combined With PD1 mAb for Relapsed or Refractory Diffuse Large B Cell Lymphoma
Phase: PHASE1
Conditions: Diffuse Large B-cell Lymphoma
Sponsor: Second Affiliated Hospital, School of Medicine, Zhejiang University

CD19 CAR-T Expressing IL7 and CCL19 Combined With PD1 mAb 
for Relapsed or Refractory Diffuse Large B Cell Lymphoma 
 
 
 
 
Protocol Number:  
 
NHL-7×19-2020-01 
Study Center: 
2nd Affiliated Hospital, School of Medicine, 
Zhejiang University 
 
 
 
 
 
 
 
 
 
Version:             1.0 
Date:               March 20, 2020 
 
 

Overview 
Name 
 
Phase Ib clinical study of CD19 CAR-T expressing IL7 
and CCL19 combined with PD1 mAb in the treatment 
of relapsed/refractory diffuse large B cell lymphoma 
Purpose 
 
To evaluate the safety and efficacy of CD19 CAR-T 
(hereinafter referred to as CD19-7×19 CAR-T) 
expressing IL7 and CCL19 combined with PD1 mAb in 
the treatment of relapsed / refractory diffuse large B-
cell lymphoma, and to provide a new method for the 
treatment of relapsed / refractory diffuse large B-cell 
lymphoma. 
Sponsor 
2nd Affiliated Hospital, School of Medicine, Zhejiang 
University 
Indication 
relapsed / refractory diffuse large B-cell lymphoma 
Study Design To evaluate the efficacy and safety of CD19-7×19 
CAR-T combined with PD1 mAb in the treatment of 
relapsed / refractory large B cell lymphoma. 
 
 
Subjects 
 
Twenty four patients aged 18-75 years were recruited, 
including 9 patients in the dose increasing stage and 15 
patients in the dose expanding stage. If the number of 
patients in Ib phase increased, the number of patients in 
the expanding stage decreased accordingly. Patients 

met the inclusion / exclusion criteria. Considering the 
failure of cell preparation, or the inability of cell 
transfusion due to disease progression during cell 
preparation, the number of participants in cell 
preparation in this study may be more than the number 
of planned cases. 
Study Period 24 months 
 
 
 
 
 
Selection 
Criteria 
 
Inclusion Criteria: 
(1) Age ≥ 18, upper limit 75, unlimited for men and 
women; 
(2) ECOG score 0-3; 
(3) Histologically confirmed diffuse large B-cell 
lymphoma (DLBCL) [according to who 2008]; 
(4) CD19 was positive (immunohistochemistry or flow 
cytometry). 
(5) The definition of refractory or relapse of DLBCL is: 
no complete remission after 2-line treatment; disease 
progression in any treatment process, or disease 
stabilization time equal to or less than 6 months; or 
disease progression or relapse within 12 months after 
hematopoietic stem cell transplantation; 
(6) The previous treatment of diffuse large B cell 

lymphoma must include rituximab (CD20 mAb) and 
anthracycline; 
(7) There should be at least one measurable focus. It is 
required that any length of lymph node focus should be 
greater than 1.5cm or any length of extranodal focus 
should be greater than 1.0cm. PET-CT scan focuses 
should have uptake (SUV is greater than liver blood 
pool); 
(8) The absolute value of neutrophils in peripheral 
blood ≥ 1000 / μ L, platelet ≥ 45000 / μ L; 
(9) Heart, liver and kidney function: creatinine < 
1.5mg/dl; ALT (alanine aminotransferase) / AST 
(aspartate aminotransferase) 2.5 times lower than the 
normal upper limit; total bilirubin < 1.5mg/dl; heart 
ejection fraction (EF) ≥ 50%; 
(10) Sufficient understanding ability and voluntary 
signing of informed consent; 
(11) Those with fertility must be willing to use 
contraceptive methods; 
(12) According to the judgment of the researchers, the 
expected survival time is more than 4 months; 
(13) Willing to follow visit schedule, administration 

plan, laboratory inspection and other test steps. 
Exclusion Criteria: 
(1) History of other tumors; 
(2) Hematopoietic stem cell transplantation was 
performed within 6 weeks; 
(3) Any target car-t treatment was performed within 3 
months before the car-t treatment; 
(4) Previous use of any commercially available PD-1 
mAb; 
(5) Cytotoxic drugs, glucocorticoids and other targeted 
drugs were received within 2 weeks before cell 
collection; 
(6) Active autoimmune diseases; 
(7) Uncontrollable infection of active bacteria and 
fungi; 
(8) HIV infection, syphilis infection; active hepatitis B 
or C: hepatitis B: HBV-DNA ≥ 1000IU / ml; hepatitis 
C: HCV RNA positive and liver function abnormal. 
(9) Known central nervous system lymphoma. 
 
 
 
This study is a single arm, single center, open 
phase I B clinical study. The study will be divided into 
two phases: 

 
 
Trial 
group 
and treatment 
plan 
Stage I is a dose increasing study. Nine effective 
subjects are planned to be recruited to complete cell 
reinfusion. The efficacy and safety of cd19-7 × 19 car-t 
combined with PD1 antibody in the treatment of 
relapsed / refractory B cell tumors are preliminarily 
evaluated. 
Stage II is the extended study stage. According to 
the data of safety tolerance, proliferation and survival 
of cd19-7×19 CAR-T in vivo, clinical efficacy and 
other data obtained in stage I, the recommended dose 
for 
entering 
this 
stage 
is 
determined 
after 
comprehensive consideration, and 15 effective subjects 
are recruited to further evaluate its effectiveness and 
safety. 
In the dose increasing stage, the last subject in each 
dose group can enter the next dose group after 
completing the dose limiting toxicity (DLI) evaluation 
window 30 days after reinfusion and 14 days after the 
application of PD-1 monoclonal antibody, and after the 
safety supervision committee combined with the 
clinical safety and pharmacokinetic data evaluation 
consent. 

In the extended study phase, the dose is selected 
by the PI according to the first phase and reported to the 
Ethics Committee (Safety Supervision Committee) for 
approval. 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment 
process 
Preparation of test preparation: preparation of CD19-
7×19 CAR-T cells 
In the experimental group, blood cells were separated 
and PBMCs were separated by Ficoll lymphocyte 
separation technique. The cell density of 2 × 106 PBMC 
/ ml was inoculated into the cell culture flask. 
According to the ratio of beads: cell = 3:1, the pre 
washed human t-activator CD3 / CD28 Dynabeads 
(cat#11161D, life technologies) were added to activate 
T cells. After 24 hours, the cell density was 
concentrated to 3-5 × 106 cells / ml, and the lentivirus 
vector carrying CD19-7×19 CAR was infected in a 
volume ratio of 1:10. At the same time, the transfection 
reagents Polybrene (4ug/ml) and IL-2 (300IU/ml) were 
added. After 4 hours, the fresh medium was 
supplemented and the cell density was adjusted to 
1×106cell/ml. On the second day, the cells were 
centrifuged to remove the uninfected virus solution and 

replaced with fresh culture medium. After that, the cells 
were replaced with fresh culture medium every 2-3 days 
according to the growth of cells. The transfection 
efficiency of CD19-7×19 CAR was measured by 0.5-1 
× 106 cells on the 4th-5th day. After 12-14 days of 
culture, cells were harvested to prepare 300ml cells for 
reinfusion. 
CD19-7 × 19 car-t cell detection index: 
① Cell number (number of CD19 positive car cells): 
Target dose: 1×106/kg, 2×106/kg, 3×106/kg (allowable 
fluctuation range: ± 20%). 
② Cell morphology: the surface of microscope cell is 
smooth; 
③ Cell viability: more than 90%; 
④ Cell surface markers: CD3 +, CD8 +, CD107a +, 
Car19 +, CD45RA, CCR7;  
⑤CD19-7×19 CAR transfusion method: 300ml of cell 
suspension product (CD19-7×19 CAR-T+normal saline 
+ 2% human albumin) was infused back to the patient 
by intravenous infusion. 
Dosage of the test preparation: CD19-7×19 CAR-T 
treatment is divided into two stages, stage I is divided 

into three dose ramps: 1×106/kg, 2×106/kg, 3×106/kg 
car to transduce auto T cells; stage II is determined by 
the researcher based on safety tolerance, expansion and 
duration of CD19-7×19 CAR-T in vivo, clinical 
efficacy and previous clinical research experience. On 
day 0, an intravenous transfusion was performed. 
FC pretreatment scheme: 30mg/m2/day, 3 days (D-5, -
4, -3); CTX 500 mg/m2, 3 days (D-5, -4, -3). See section 
3.9 for chemotherapy plan. 
Dosage and usage of PD1 mAb: the dosage is 200mg / 
time, and it is used for three weeks for six consecutive 
courses (PD-1 can be stopped for those who are 
assessed as disease progression for three months). 
 
 
 
 
Program 
 
At the specific time points specified in the 
evaluation plan, subjects will be subject to the 
following procedures: 
(1) Obtain informed consent, general medical 
history (including previous treatment of 
lymphoma), physical examination (including vital 
signs and physical condition), neurological 
evaluation, blood routine test, biochemical test, 

cytokine, C-reactive protein, lymphocyte subtype 
and other tests. 
(2) Women with fertility will be tested for 
pregnancy in urine or serum. 
(3) Subjects will also receive baseline 
electrocardiogram (ECG), echocardiography (echo), 
brain magnetic resonance imaging (MRI), B 
ultrasound, positron emission tomography - 
computed tomography (PET-CT). 
(4) The patients were assessed to complete 
leukocyte separation. 
(5) Pre treatment of FC scheme shall be carried out 
within - 5 to - 3 days after car-t back transmission. 
(6) CD19-7×19 CAR-T infusion. 
(7) On the 31st day of CD19-7×19 CAR-T infusion, 
PD1 MAb (baizean mAb) was infused 
intravenously. 
Subjects were regularly asked to report concomitant 
medication and adverse events and to assess their 
disease throughout the study. 

 
 
 
 
Observation 
index 
1. Primary research objectives: 
(1) To evaluate the safety of CD19-7×19 CAR-T cells 
combined with PD1 mAb in the treatment of relapsed 
or refractory diffuse large B cell lymphoma; 
(2) Objective remission rate (CR + PR) of CD19-7×19 
CAR-T cells combined with PD1 mAb in the treatment 
of recurrent or refractory diffuse large B-cell lymphoma 
was evaluated. 
2. Secondary research objectives: 
(1) 2-year PFS, relapse rate, OS and dor; 
(2) After treatment, biological markers such as car T 
cells and cytokines were monitored; 
(3) To study the relationship between the efficacy and 
side effects of different car-t cell dosage groups and 
PD1 mAb combination. 
Safety 
analysis 
Vital signs, adverse events, changes in clinical 
laboratory data and early withdrawal, etc. 
 
 
 
 
 
1. Selection of statistical analysis data 
(1) Fas (full analysis set): according to the principle of 
intent to treatment (ITT), all patients will enter the full 
analysis set of this trial. For patients who withdraw 
from the study in advance for various reasons, the 

 
 
 
 
Statistical 
analysis 
results of the corresponding evaluation points will be 
filled with the last observation carry forward (LOCF). 
(2) Per protocol set (PPS): all of them met the entry 
conditions to enter the study and complete the 
treatment, with good compliance. 
(3) Safety analysis population: defined as patients 
receiving car-t cell infusion and patients receiving a 
course of post-treatment safety evaluation of PD-1 
mAb constitute the safety analysis set of this study. 
(4) Subgroup analysis: the safety and efficacy of car-t 
cells in different dose groups and in the groups 
receiving 1-6 times of PD-1 mAb were analyzed. 
(5) Intermediate analysis sets: DLT evaluation is 
applicable to patients with IB dose climbing. DLT 
evaluation time: 30 days after car-t infusion and 15 
days after the first treatment with PD-1 mAb. Mid 
term evaluation patients: all patients in IB stage were 
enrolled, and the efficacy evaluation was completed 
for 3 months. 
2. Analysis plan 
According to the research plan, biostatisticians and 
main researchers make statistical analysis plan, and 

complete and form documents before data locking. 
The statistical analysis software applies SAS statistical 
analysis software 9.1.1. 
(1) Efficacy evaluation 
The measurement data were expressed by X ± s, and 
the comparison of different time points was analyzed 
by repeated measurement analysis of variance. P < 
0.05 was statistically significant. Kaplan Meier 
survival curve analysis was used for survival analysis 
to calculate the median survival time and progression 
free survival time. 
(2) Safety evaluation 
The safety evaluation will be descriptive, 
including adverse events, laboratory examination 
indicators, vital signs, etc. The type, severity, 
frequency and relationship with the study drug of all 
adverse events occurred during the study will be 
described in a list. The suspension of the study due to 
adverse events and cases of serious adverse events will 
be specially noted. For laboratory examination, all 
completed examination items are listed in the form of 
a cross table before and after treatment (according to 

the judgment of the clinician). Items with abnormal 
values and clinical significance need to be listed. 
 
 
 
Dose limited 
toxicity (DLT) 
Dose limiting toxicity (DLT) is defined as: 
CD19-7×19 CAR-T cell infusion within 30 days is 
likely or definitely related to any of the following 
conditions: 
(1) Grade 3-5 anaphylaxis; 
(2) In addition to B-cell clearance, the level of CTCAE 
was 2-5; 
(3) The non hematologic toxicity of any major organ ≥ 
Level 3 was not relieved (reduced to level 3 toxicity) or 
lasted for more than 28 days after intravenous 
administration of dexamethasone 10mg / 12h for 7 days 
(or the same dose of corticosteroids) or trozumab 
8mg/kg for 3 times. 
 
Maximum 
tolerable dose 
(MTD) 
The incidence of dose limited toxicity (DLT) was lower 
than the highest dose level of 33% (at least 6 subjects 
were required to accept this dose level to evaluate the 
toxicity).