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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                                                  |
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| Study Center:      | 2nd Affiliated Hospital, School of Medicine,  Zhejiang University |  
| Version:   | 1.0   |                |
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| 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                                                                                                    |
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| 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 Bcell 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. Twenty four patients aged 18-75 years were recruited,  including 9 patients in the dose increasing stage and 15 |
| Subjects        | 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.   |                                                                                                                                                                                                                                                                                                                                                                                                                                               |
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| Study Period                                                                                                                                                                                                                                                                                    | 24 months 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                                                                                                                                                                                                                                                                               |
| Selection                                                                                                                                                                                                                                                                                       | lymphoma (DLBCL) [according to who 2008];                                                                                                                                                                                                                                                                                                                                                                                                     |
| Criteria                                                                                                                                                                                                                                                                                        | (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   |
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| plan, laboratory inspection and other test steps. Exclusion Criteria: (1) History of other tumors; (2) Hematopoietic stem cell transplantation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | was   |
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| 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: |       |  
| Stage I is a dose increasing study. Nine effective    |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                          |
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| subjects are planned to be recruited to complete cell |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                          |
| Trial                                                 | group                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | reinfusion. The efficacy and safety of cd19-7 × 19 car-t |
| and treatment                                         | combined with PD1 antibody in the treatment of  relapsed / refractory B cell tumors are preliminarily                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                          |
| plan                                                  | 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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                |
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| by the PI according to the first phase and reported to the  Ethics Committee (Safety Supervision Committee) for  approval. 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 |                                                                                                                                                                                                                                                                |
| Treatment                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | volume ratio of 1:10. At the same time, the transfection                                                                                                                                                                                                       |
| process                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | 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  1×106 cell/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   |
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| 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). At the specific time points specified in the  evaluation plan, subjects will be subject to the  following procedures: (1) Obtain informed consent, general medical   |                                                                                                                                                                                            |
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| Program                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | 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.   |
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| 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   |                                                                                                                                                                                                                                                                                                                                                                                                                                          |        |         |         |         |    |          |
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| Observation  index                                                                                                                                                                                                                          | 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                                                                                                                                                                                                                                      | Vital                                                                                                                                                                                                                                                                                                                                                                                                                                    | signs, | adverse | events, | changes | in | clinical |
| analysis                                                                                                                                                                                                                                    | 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                                                                                                                  |        |         |         |         |    |          |  
| 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.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
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| Statistical  analysis                                                                                                                                                                                                                                    | (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   |
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| the judgment of the clinician). Items with abnormal  values and clinical significance need to be listed. 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   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |             |
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| Dose                                                                                                                                                                                                                                                         | limited                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | conditions: |
| toxicity (DLT)                                                                                                                                                                                                                                               | (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. The incidence of dose limited toxicity (DLT) was lower  than the highest dose level of 33% (at least 6 subjects |             |
| Maximum  tolerable dose                                                                                                                                                                                                                                      | were required to accept this dose level to evaluate the                                                                                                                                                                                                                                                                                                                                                                                                                                                      |             |
| (MTD)                                                                                                                                                                                                                                                        | toxicity).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |             |