figure 1 study schema
5.4.6 day +1 through day

Patients will be seen daily through day+7 of infusion. This will occur either in the inpatient setting or with daily visits to the Day Hospital through day+7. At these evaluations, the following will occur:  
- Physical Examination including weight, concomitant medications, and review of adverse events.  
- Vital signs: Blood Pressure, Temperature, Respiratory rate, Pulse & Pulse Ox.  
- CBC with differential
- Comprehensive Metabolic Panel: Albumin, Alkaline Phosphatase, AST, Bicarb, Bili T,
Calcium (total), Chloride, Creatinine, Glucose, Potassium (K), Sodium (NA), Protein Total, BUN, and ALT.  
- Uric Acid
- LDH
- Coagulation Assessments: PT/INR, PTT, Fibrinogen, and D-Dimer. - Ferritin/CRP/ESR
- Research Studies as per schedule of events (Table 1)
o T-Cell Persistent Studies—Will not be repeated once there are two consecutive negative results.  
o Cytokine Studies o Neurological assessments o T-cell repertoire diversity testing from peripheral blood
figure 1 study schema
5.4.7 follow-up visits (day+10 to

Patients with an ongoing response will be followed for up to 2 years after CAR-20/19-T cells infusion as per this protocol. After Day 28, testing can be performed at the local oncologist office and results sent to MCW/FH other than dates mentioned in the study calendar. The following procedures will be performed at specified intervals, **see Table 1 per schedule of**
events.  
- Physical Examination, including weight, Performance Status Assessment (Karnofsky score), concomitant medications, and review of adverse events.  
- Vital signs: Blood Pressure, Temperature, Respiratory rate, Pulse & Pulse Ox
- CBC with differential
- Comprehensive Metabolic Panel: Albumin, Alkaline Phosphatase, AST, Bicarb, Bili T,
Calcium (total), Chloride, Creatinine, Glucose, Potassium (K), Sodium (NA), Protein Total, BUN, and ALT.
- Uric Acid - LDH - Coagulation Assessments: PT/INR, PTT, Fibrinogen, and D-Dimer - Lipid Panel
- Thyroid Function Tests: TSH, Free T4 - Quantitative Immunoglobulins (IgM, IgG, IgA) - Urinalysis
- Bone Marrow Transplant markers
- Beta-2-Microglobulin
- Ferritin/ESR/CRP
- Radiologic imaging:
o CT neck/chest/abd/pelvis or PET/CT skull base to mid-thigh o At 1 month, 6 months, and 1 year. From 1 year to 2 years, scans will be done yearly.  
a) Scans would only be completed in follow-up only for patients who remain in remission since entering the study. Patients with progressive disease will not require any further imaging.  
- Bone Marrow Biopsy and aspirate as per Table 1 - Electrocardiogram (ECG)
- Research Studies:
o T-Cell Persistent Studies—Will not be repeated once there are two consecutive negative results.  
o Q-PCR for Integrated Vector DNA—Will not be repeated once there are two consecutive negative results.  
o Detectable RCL by Q-PCR for VSV-G—Will not be repeated once Q-PCR for integrated vector DNA is negative at two consecutive timepoints.  
o Cytokine Studies o Neurological assessments o T-cell repertoire diversity testing from peripheral blood o Quality of Life Studies o Tryptophan Metabolites o PaxGene RNA gene expression VISITS REQUIRED AT MCW/FH: Beyond Day +28, patients will have required visits at MCW/FH on Day +90 (+/- 7 days), Day+180 (+/- 14 days), Day 365 (+/- 1 month), +1.5 years (+/- 1 month) and 2 years (+/- 1 month). For timepoints not falling on those dates, local oncology evaluation will suffice and records will be obtained accordingly.
figure 1 study schema
progressive disease

For patients who develop **progressive disease** will not follow the above study calendar and will placed in a follow-up plan as detailed below with visit with their local oncologist every 6 months starting from CAR-T infusion date for up to 2 years and if alive will subsequently roll into our long-term follow-up protocol. At these visits the following should be performed
- Physical Examination, including vitals - Survival/Progression status
- Subsequent Treatments Post-CAR-T infusion
- Targeted concomitant medications o Including IVIG, immunosuppressive medications, prophylactic anti-infective medications
- Targeted adverse events recording o Including New malignancies, new incidence or exacerbation of pre-existing neurologic disorder, new incidence or exacerbation of prior rheumatologic disorder, or other autoimmune condition, and new incidence of hematologic disorder
- CBC with differential
- Comprehensive Metabolic Panel: Albumin, Alkaline Phosphatase, AST, Bicarb, Bili T,
Calcium (total), Chloride, Creatinine, Glucose, Potassium (K), Sodium (NA), Protein Total, BUN, and ALT.  
- Quantitative Immunoglobulins (IgM, IgG, IgA) - Absolute B and T cell lymphocyte counts
- Research Studies:
o Patients will be required to have blood sent to MCW (See **Appendix K** for instructions on sending samples to MCW) or return to MCW for testing at the following timepoints until testing is negative for persistence of CAR-T cells: +6 months, +1 year, +1.5 years, and +2 years post-CAR-T infusion and will include the following listed below. Once there are TWO negative results, no further visits to MCW or blood samples to MCW will be mandated:
a) T-Cell Persistent Studies—Will not be repeated once there are two consecutive negative results. Additional samples (blood, CSF, ascites, tissue, etc.) for T-cell persistence can be performed per the investigators discretion as felt to be clinically indicated.  
b) Q-PCR for Integrated Vector DNA—Will not be repeated once there are two consecutive negative results.  
c) Detectable RCL by Q-PCR for VSV-G—Will not be repeated once Q-PCR
for integrated vector DNA is negative at two consecutive timepoints.
figure 1 study schema
5.4.8 long-term/survival follow-up procedures

Patients will be followed for up to 2 years after CAR-20/19-T cells infusion or until progression.  
Patients who progress will be followed for survival, progression, persistence of CAR-T cells, and future treatments. Patients who progress post treatment will not have any further required disease assessment on study, and management of their disease will return to their treating physician. They will be required to return for other study laboratory testing and evaluations as mandated per the protocol as outlined above. Patients who are alive beyond 2 years will be placed on a long-term follow-up protocol for a total of up to 15 years for monitoring of postgenetically modified therapies.
figure 1 study schema
5.5 study withdrawal procedures

Patient-Initiated Withdrawal: A patient may decide to withdraw from the study at any time.  
Investigator-Initiated Withdrawal: The Investigator will withdraw a patient whenever continued participation is no longer in the patient's best interests. Reasons for withdrawing a patient include, but are not limited to, disease progression, the occurrence of an adverse event or a concurrent illness that prevents further administration of treatment, a patient's request to end participation, a patient's noncompliance or simply significant uncertainty on the part of the Investigator that continued participation is prudent. There may also be administrative reasons to terminate participation, such as concern about a patient's compliance with the prescribed treatment regimen.  
Withdrawal Documentation Procedure: The reason for study withdrawal and the date the patient was removed from the study must be documented in the case report form.
figure 1 study schema
6 treatment plan 6.1 lymphodepletion

Patients will receive lymphodepleting conditioning regimen prior to CAR-20/19-T cells starting on Day -4. Lymphodepletion will start based on the planned date of infusion of CAR-20/19-T cells. Tumor lysis prevention with allopurinol daily (dose per institutional standards) will be initiated the day of chemotherapy. Levetiracetam (Keppra) prophylaxis dosed per institutional standards will be initiated in all patients to prevent possible neurologic toxicity. Anti-emetics and steroids can be provided pre-treatment as per standard of care protocols for these treatment regimens. Please see Appendix E for guidelines for CAR-T cell infusion. The lymphodepletion regimens is as follows:
figure 1 study schema
fludarabine 30 mg/m2 on day

This regimen is based on published lymphodepletion protocols for adult patients receiving CD19 CAR-T cells. Fludarabine and Cyclophosphamide combination is the most utilized lymphodepletion regimen prior to CAR-T cell therapy and the above regimen was presented at ASH 2016 and used in a large cohort of >100 patients with published safety and toxicity data.  
[41, 48] Fludarabine and Cyclophosphamide dosing will be based on actual body weight. For patients who are neutropenic (ANC<1000 k/µL) at the start of lymphodepletion due to underlying hematologic disease or as a result of prior chemotherapy related toxicity, cyclophosphamide will be omitted from the lymphodepletion regimen.  
Phase 1 Dose Escalation/Expansion: CAR-20/19-T cells will be administered per actual body weight in the lymphopenic phase on Day 0 & Day 1. Dose will be maxed at a weight of 80 kilograms. Phase 1b Cohort : CAR-20/19-T cells will be administered as a single dose on Day 0. Dose will be maxed at a weight of 80 kilograms
figure 1 study schema
6.2 preparation of car-20/19-t cell

Cell Manufacturing The MNC, Apheresis product obtained from the patient on Day -15 will be washed free of platelets and plasma and the CD4+ and CD8+ T cells will be purified from the MNC, activated through the CD3 and CD28 antigens, then transduced with the CD20/CD19 CD3ζ/41BB
lentiviral vector. The cells will be expanded *in vitro,* harvested from culture, and then administered within 48 hours of production in an isotonic electrolyte solution (Plasma-Lyte® A or Normosol® R) containing 2.5% HSA or cryopreserved for administration in a freezing solution of Plasmalyte A or Normosol R containing 10% DMSO and 4% HSA. Testing will be performed at two mandatory time points (in process day-6 and at harvest) and will include the following (Please see **Appendix D** for details on testing, including those tests for which results are required for product release):
figure 1 study schema
final product testing (harvest)

- Sterility o endotoxin o mycoplasma o bacterial and fungal cultures
- Phenotype o CD3, CD4, CD8, central memory and effector memory T cell subsets, o NK cells at harvest o NK-T cells at harvest o B cells at harvest o T regs at harvest
- Viability and viable cell numbers - Effector activity (lysis and cytokine product in response to B cell targets)
- Copy number (qPCR) at harvest
- VSVg (qPCR) at harvest
- % transduced cells (light chain expressing T cells)
Cells will be harvested on Day 14 of production for fresh administration or cryopreserved for later infusion **(Appendix D)**. The day cells are ultimately administered will be deemed Day 0 for study calendar and schedule of activities. All testing will be performed in accordance with FACT
and FDA regulations. The entire manufacturing process up until removal from culture will be performed by a computer- controlled program using the CliniMACS Prodigy device.
figure 1 study schema
cell thawing (if indicated)

For patients who require cryopreservation the following will be performed: The frozen cells will be thawed in the cell processing lab and transported by laboratory staff to the subject's bedside either at the inpatient oncology unit of FH or the outpatient day hospital under the care of a Hematology/Oncology attending. If the CAR-20/19-T cell product appears to have a damaged or leaking bag, or otherwise appears to be compromised, it should not be infused, and should be returned to production laboratory.
figure 1 study schema
positive sterility test (after infusion)

In the scenario that CAR-T cells are administered prior to completion of the 14 days of sterility testing, it is possible that a sterility test returns positive after administration. In that scenario, we will follow our standard operating procedure for positive cultures after infusion of cell therapy products. In general, this involves the following:
1. Cell processing lab will contact the PI on the study at the time of this finding. 2. The PI will discuss these findings with the patient 3. The PI will order appropriate infectious work-up and provide antimicrobial coverage as indicated based on the results of the sterility tests. 4. Reporting—the PI on the clinical trial will report this to the DSMC, IRB, and the FDA
figure 1 study schema
6.3 administration of car-20/19-t cell

Patients will receive the infusion of the CAR-20/19-T cells as an inpatient on the oncology unit or in the outpatient Day Hospital under the clinical care of hematology/oncology attending. Patients will receive institutional standard for infusion reaction prophylaxis (e.g., Acetaminophen and diphenhydramine hydrochloride, or other acceptable alternatives, as pre-treatment).  
Patients will be on allopurinol for tumor lysis prevention and levetiracetam (Keppra) with initiation of lymphodepleting chemotherapy **(See Appendix E for details)**. Pretreatment with steroids is contraindicated. Patients will receive CAR-20/19-T cells by IV infusion via central line for a total viable, transduced, cell dose ranging from 1.0 x 105 cells/kg to 2.5 x 106 cells/kg based on phase of study. The dose of cells may be limited if calculations performed at the time of administration or cryopreservation indicates that the patient would receive > 1.8 x 108 nonviable cells/kg. Patients who fail to meet the target cell dose will be allowed infusion and will be followed for toxicity and adverse events as all other patients. The infusions will occur approximately 1-7 days following chemotherapy.
figure 1 study schema
premedication

Potential side effects following T cell infusions include transient fever, chills, and/or nausea. The subject will be pre-medicated with acetaminophen and/or diphenhydramine hydrochloride as per institutional standards prior to the infusion of CAR-20/19-T cells. These medications may be repeated every six hours as needed. Epinephrine will be readily available in the rare scenario of an allergic reaction. A course of non-steroidal anti-inflammatory medication may be prescribed if the patient continues to have fever not relieved by acetaminophen. It is recommended that patients not receive systemic corticosteroids such as hydrocortisone, prednisone, prednisolone (Solu-Medrol) or dexamethasone (Decadron) at any time, except in the case of a life-threatening emergency, since this may have an adverse effect on T cells. If corticosteroids are required for an acute infusional reaction, an initial dose of hydrocortisone 100 mg is recommended.
figure 1 study schema
prophylaxis therapies

Patients will receive appropriate prophylaxis with CAR-T cell therapy as recommended in Appendix E. Briefly levetiracetam will be given for seizure prophylaxis, institutional standards will be applied for pneumocystis pneumonia (PCP), viral, and neutropenic prophylaxis.
figure 1 study schema
infusion

On the day of the first infusion (Day 0), patients will have a CBC with differential, and assessment of T-cell subsets prior to administration of CAR-20/19-T cells since chemotherapy is given in part to induce lymphopenia.  
The CAR-20/19-T cell dose will be administered by IV infusion. Dosing will be split during the phase 1 portion of the trial, with 30% given on day 0, and 70% given on day 1. If there is no significant infusional toxicity, in the phase 1b portion of the trial cells will be administered as a single dose on Day 0. As detailed above, the cells will be administered fresh or thawed if indicated in the LPL and placed in a validated transport container at 1-10oC and brought directly to the patient's bedside. If the CAR-20/19-T cell product appears damaged or leaking, or otherwise appears to be compromised, it should not be infused, and should be returned to production laboratory. The infusion will take place in the patient room on the oncology inpatient units or in the outpatient day hospital BMT rooms, using precautions for immunosuppressed patients. The transduced T cells will be administered by intravenous infusion. The infusion nurse spikes HPC product bag and administers through blood tubing accordingly. The duration of the infusion will be approximately 5-15 minutes. Each infusion bag will have affixed to it a label containing the following: "FOR AUTOLOGOUS USE ONLY". In addition, the label will have at least two unique patient identifiers such as the subject's initials, birth date, and study number. Prior to the infusion, two individuals will independently verify all this information in the presence of the subject and so confirm that the information is correctly matched to the participant. Subjects will be infused and premedicated as described. Subjects' vital signs (temperature, respiration rate, pulse, and blood pressure, pulse oximetry) will be done prior to dosing, at the end of the infusion and every 15 minutes thereafter for a minimum of 1 hour. A blood sample for determination of a baseline CAR-20/19-T cell level is obtained any time prior to the infusion and Day+1 (and sent to the LPL Labs) and then will be completed as per the study calendar. Emergency medical equipment (i.e., emergency trolley) will be available during the infusion in case the subject has an allergic response, or severe hypotensive crisis, or any other reaction to the infusion. Patients experiencing toxicities from their preceding cytoreductive chemotherapy will have their infusion schedule delayed until these toxicities have resolved. The specific toxicities warranting delay of T cell infusions include:
1) Pulmonary: Requirement for supplemental oxygen >2L by nasal cannula to keep saturation greater than 95% or presence of radiographic abnormalities on chest x-ray that are progressive; 2) Cardiac: New cardiac arrhythmia not controlled with medical management 3) Hypotension: requiring pressor support 4) Active Infection: Fever>101 F and/or positive cultures for bacteria, fungus, or virus. Patients who are unable to receive the CAR-20/19-T cells within 7 days of completion of chemotherapy will be removed from the protocol.
figure 1 study schema
febrile reaction

In the unlikely event that the subject develops sepsis or systemic bacteremia following CAR-Tcell infusion, appropriate cultures and medical management should be initiated.  
| Table 2: Regimen Description Study Drug Premedication3                                                                                                                                                                                                                                                                                                                                                                            | Dose           |                              | Route   | Schedule      | Cycle  Length   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|------------------------------|---------|---------------|-----------------|
| CAR-20/19-T cells1                                                                                                                                                                                                                                                                                                                                                                                                                | Tylenol PO     | 1.0 x 105-2.5 x 106cells/kg2 | IV      | 30% on Day 0  |                 |
| (Phase 1)                                                                                                                                                                                                                                                                                                                                                                                                                         | Benadryl IV/PO | 70% on Day 1                 | 28 days |               |                 |
| CAR-20/19-T cells                                                                                                                                                                                                                                                                                                                                                                                                                 | As above       | 2.5 x 106 cells/kg2          | IV      | 100% on Day 0 |                 |
| (Phase 1b)                                                                                                                                                                                                                                                                                                                                                                                                                        |                | 28 days                      |         |               |                 |
|                                                                                                                                                                                                                                                                                                                                                                                                                                   |                |                              |         |               |                 |
| 1Patients with ≥ grade 2 non-hematologic CTCAE toxicity will have the 2nd dose held up to 7 days until  symptoms resolve or will not be administered at the discretion of the PI 2Cell number include only viable T cells expressing the CAR as determined by flow cytometry. Cell dose maxed  at 80 kg or if the infusion contains >1.8 x 108/kg non-viable cells. 3Recommended premedication, dosed per institutional standards |                |                              |         |               |                 |
figure 1 study schema
6.4 dose escalation schedule

For this phase 1/1b study we plan to include multiple histologies including CLL/SLL and B-cell NHL. Based on previous CAR-T studies (as outlined in Section 1.6) our goal treatment dose is 2.5 x 106 cells/kg. Due to concern that a dual CAR-T cell may increase risk of CRS our starting dose for this study will be 2.5 x 105 cells/kg (1 log reduction) and we will escalate based on DLTs to our goal dose. We will utilize a 3+3 escalation design for this study **(Figure 9)**. *At each* dose level, the first two patients will be monitored for a full 28 days after CAR-T infusion prior to subsequent patient's treatment with CAR-T cells to allow monitoring of acute and sub-acute toxicities. After that patients will be *infused no sooner than at 14-day intervals among the* specified dose level or in-between two dose levels. The first three patients with NHL or CLL/SLL
will be treated with 2.5 x 105 cells/kg. If 0/3 patients experience a DLT, the dose will increase to 7.5 x 105 cells/kg. If 1/3 patients at the 2.5 x 105 cells/kg experiences a toxicity, then an additional three patients will be treated at this dose level. If ≤1/6 patients have a DLT at this level, the dose will increase to 7.5 x 105 cells/kg. If ≥2/6 patients have a DLT at 2.5 x 105 cells/kg, the dose will de-escalate to 1.0 x 105 cells/kg. If 2/3 patients in our first cohort have DLTs, the dose will also de-escalate to 1.0 x 105 cells/kg. If at the 1.0 x 105 cells/kg there are ≥2 DLTs out of a maximum of 6 patients, the study will close due to excessive toxicity. If there is 01 DLTs in 3-6 patients, the 1.0 x 105 cells/kg dose will be selected for expansion cohort (**Figure**
10).  
We will utilize a similar dosing schema should we escalate to the 7.5 x 105 cells/kg dose. If there are 0-1 toxicities out of 3-6 patients, the dose will escalate to our goal dose of 2.5 x 106 cells/kg. If there are ≥2 toxicities in 3-6 patients at 7.5 x 105 dose, then the 2.5 x 105 cells/kg dose will be selected for our expansion cohort. At our goal dose level (2.5 x 106 cells/kg) if there is 0-1 DLTs out of 3-6 patients, this dose will be selected for expansion. If there are ≥2 toxicities in 3-6 patients at the 2.5 x 106 cells/kg dose, then the 7.5 x 105 cells/kg dose will be selected.  
Once a dose has been selected within the NHL and CLL/SLL cohort, this dose will be utilized for the Dose Expansion phase of the study (Section 6.5). The dose expansion cohort will proceed only after approval from the local IRB/DSMC and the FDA.  
| Table 3: Dose Escalation CAR-T cells Dose Level Dose of Study Drug*   | Minimum # of Patients/Dose  Level   |    |
|-----------------------------------------------------------------------|-------------------------------------|----|
| -1                                                                    | 1.0 x 105 cells/kg                  | 2  |
| 0                                                                     | 2.5 x 105  cells/kg                 | 2  |
| 1                                                                     | 7.5 x 105 cells/kg                  | 2  |
| 2                                                                     | 2.5 x 106 cells/kg (goal cell dose) | 2  |
figure 1 study schema
patients who fail to produce

It is possible that some patients fail to make the targeted cell dose as required per our dose escalation/expansion schema (**Figures 9 & 10**). For those patients, we will allow administration of CAR-20/19-T cells but these patients must be replaced on the study for the purposes of our Phase 1/1b study. Despite not achieving the desired dose level there are still potential toxicities associated with CAR-20/19-T cell administration and so these patients will undergo identical study procedures and will be followed for DLTs, AEs, and survival as all other patients. For this cohort of patients who receive a non-specified dose, if there is ≥33% rate of DLT as defined in section 3.4, then future patients who fail to make the desired dose level will no longer be allowed to be treated in this manner. Standard operating procedures for patients treated at a non-specified cell dose are listed in **Appendix H**.  
Figure 9: Dose Escalation Phase
figure 1 study schema
6.5 dose expansion phase figure

Once a dose has been identified in our Dose Escalation phase we will perform a Dose Expansion at that dose level (termed Dose Level X). We will evaluate an additional 6 patients in NHL/CLL at this Dose Level X **(Figure 10).** In this dose expansion phase is there are ≥2 patients with DLTs, no further patients will be allowed enrollment. The dose expansion phase will commence after identification of a dose level in the dose escalation cohort and after approval from both internal and external monitoring agencies (DSMB/IRB/FDA).
figure 1 study schema
6.6 phase 1b (single dose

Once the final patient on the Phase 1 protocol has enrolled on the trial, we will initiate enrollment on the Phase 1b portion to test the safety of single dose administration compared to fractionated dosing. This phase will include a 9 patient expansion at 2.5 x 106 cells/kg CAR-T
cells administered in a single dose (rather than split dose). The first two patients at this dose level will be admitted to the hospital and monitored for 14-days prior to subsequent infusion to ensure there are not unexpected toxicities associated with single-dosing. If there are no DLTs in these first two patients, then additional patients can receive cells either outpatient or inpatient and be treated as indicated. This cohort will close if there is ≥3 DLTs among the nine treated patients.
figure 1 study schema
6.7 treatment/dose limiting toxicity

Management and dose modifications associated with the AEs are outlined in Section 7: Dosing Delays/Dose Modifications.  
Dose-limiting toxicity (DLT) will be defined as a grade 3 or 4 non-hematologic toxicity possibly, probably, or definitely related to the infusion of CAR-20/19-T cells. **See section 3.4 for a** detailed definition. The dose limiting toxicity will be based on the tolerability observed during the first 28 days of treatment/observation. If multiple toxicities are seen, the presence of dose limiting toxicity should be based on the most severe toxicity experienced. Grade 3 or 4 CRS will be treated initially with IL-6 inhibition with tocilizumab and/or corticosteroids (**Appendix F**). Additional agents that can be used for refractory CRS include siltuximab or other immunosuppression at the discretion of the PI. A detailed algorithm for the management of CRS and neurotoxicity related to CAR-T cells for this study is provided in Appendix F. Supportive care measures to consider for patients receiving CAR-T cells are provided in **Appendix G**.  
As cytokine release syndrome is a new entity observed with novel cellular therapies, a modified grading system has been proposed for more appropriate assessment of dose limiting toxicities. [1, 60] We will employ this grading system to accurately grade toxicity of our CAR-20/19-T cells and a copy of the grading system can be found in **Appendix F.**
figure 1 study schema
6.8 usage of concurrent/concomitant medications

The investigational agent in this protocol is autologous CAR-20/19-T cells which as a living product will not undergo metabolism as would a pharmaceutical therapy. As such, concurrent/concomitant medications can be prescribed at the discretion of the treating physician for treatment of symptomatic conditions such as fever, nausea/vomiting, diarrhea, anxiety, etc. Antimicrobial prophylaxis can be started as per treating physician. This can include ciprofloxacin or cefepime (or other appropriate antibiotic if allergic) for antibacterial prophylaxis, acyclovir or alternative agent for antiviral prophylaxis, fluconazole (or alternative agent as indicated) for fungal prophylaxis, bactrim (or other PCP prophylaxis if sulfa allergic). Prohibited and limited therapies are detailed in the following section 6.9.
figure 1 study schema
6.9 dietary restrictions

On the inpatient service, patients will follow the FDA's Food Safety for Cancer Patient Guidelines. Otherwise no specific dietary recommendations are indicated for this study.
figure 1 study schema
6.10 prohibited medications

Patients should be off all systemic steroids, oral or IV, (not including inhaled or intranasal steroids) at the time of apheresis (for at least 7 days prior) and again starting Day 0 after administration of the CAR-20/19-T cells. Exception to this will be steroids for replacement dosing for adrenally insufficient patients or steroids for the purposes of the treatment of CRS at the discretion of the investigators listed on this protocol.  
With regards to growth factors, long-acting products such as Neulasta (pegfilgrastrim) will not be permitted starting Day -7 to Day +28. Given the potential risk of exacerbation of CRS or triggering of CRS, short-acting growth factors such as filgrastrim (neupogen) or sargramostim
(leukine) will be limited to patients with persistent neutropenia starting Day+21 provided that there are no active signs or symptoms consistent with CAR-T cell associated CRS or neurotoxicity.
figure 1 study schema
6.11 monitoring subject compliance

CAR-20/19-T cells will be administered only to eligible patients under the supervision of the investigator or identified sub-investigator(s). The appropriate study personnel will maintain records of study drug receipt and dispensing. The study agent will be given on the inpatient oncology units. There will be no self-administered agents as part of the investigational portion of this protocol.
figure 1 study schema
6.12 re-treatment

Patients who have received treatment on the CAR-20/19-T cell protocol may be retreated if they meet the same inclusion/exclusion criteria as listed in the clinical protocol and meet the following requirements:
1. Patient must be ≥100 days from first dose of CAR-20/19-T cells but ≤24 months from initial treatment.  
2. Have <1% detectable CAR-T cells by flow cytometry in the peripheral blood 3. Had demonstrated clinical benefit from the first dose (either PR or CR at Day 28 evaluation).  
4. Have a repeat biopsy that confirms either CD19 or CD20 positive disease post-CAR-T
cell relapse.  
5. Have not received another genetically modified CAR-T cell product either commercially or as part of a separate clinical trial.
Patients who are re-treated will not count towards our primary safety endpoint but AEs and testing will be monitored/performed in an identical fashion as their first infusion (see schedule of events). Research studies including t-cell repertoire studies, Paxgene RNA analysis, tryptophan metabolites, and QOL studies will not be repeated. Cells will be given as a single infusion on Day 0 and the lymphodepletion regimen outlined in this protocol will be utilized. Patients will not undergo repeat apheresis and CAR-T cell production, rather, they will receive remainder CAR-T
cells that are cryopreserved from their initial production (*see section 9.1.5 on stability data of* CAR-T cells post cryopreservation). Patients will receive no more than the goal dose of 2.5 x 10^6 cells/kg (max 80 kg). If the patient does not have that dose, they will receive the remainder of the CAR-T cells left. To ensure safety of re-treatment, DLTs through Day 28 will be monitored specific to this cohort. At first, a maximum of 3 patients will be allowed to be treated in this manner with concurrent enrollment allowed. If there is ≤1 DLT within the first three patients, further patients will be allowed re-treatment with CAR-20/19-T cells as clinical indicated and if they have met above criteria. After enrollment of the 4th patient or beyond in this cohort, if the DLT rate exceeds
≥33%, no further patients will be allowed retreatment. All patients will be followed for AEs, survival, response rates as per Table 1.
figure 1 study schema
7 dosing delays/dose modifications 7.1

Patients will receive the modified CAR-20/19-T cells in one fixed dose on Day 0 or two doses given 24 hours apart on Day 0 and Day +1 of this protocol. Patients will be either admitted to the oncology unit for this infusion or seen daily in the outpatient BMT Day hospital suite and will be under the care of a hematology/oncology attending. Criteria for infusion of cellular therapy include the following:
1) No new medical complication that would unduly increase risk of infusion 2) No requirement of supplemental oxygen >2L by nasal cannula to keep saturation >95% 3) No active cardiac arrhythmias not controlled with medical management 4) No hypotension requiring pressor support 5) Study treatment administration will not be delayed as a result of neutropenia, thrombocytopenia, or anemia, which are all potential risks of lymphodepleting chemotherapy 6) No uncontrolled active infectious complications 7) No fever>101o F in last 24 hours prior to infusion Patients who do not meet these criteria will have the first dose of their cells delayed for up to a maximum of 7 days beyond their goal treatment date (Day 0). If there is not resolution of those issues, the patients will be taken off protocol.  
For patients who received split dose cells, the second dose of cells given on Day 1, patients must meet the above same criteria. In addition, patients who develop clinically significant toxicities within 24 hours after their first infusion will not have their subsequent infusion given. These include grade 2 or higher infusion reactions, allergic responses, cytokine release syndrome, cardiac or pulmonary toxicities, or others deemed clinically significant by the study investigators. Transient grade 1 chills, nausea, vomiting and other mild expected infusionrelated toxicities that respond to usual supportive care measures will not require holding subsequent doses. Patients with fever≥101o F after the first cell dose felt to be related to CRS
will have the second dose held for up to 7 days until there is resolution. Any questions about subsequent dosing should be discussed with the Principal Investigator. The second dose can be delayed up to maximum of 7 days after the initial dose as per the discretion of the PI.  
| Table 4: Potential Dose Modifications for Dose #2 in setting of Cytokine Release Syndrome Grade of Event Management ≤ Grade 1 No change in planned 2nd infusion  Grade 2* Hold 2nd infusion until ≤ Grade 1 Resume at same dose level Grade 3 Hold 2nd infusion Grade 4 Hold 2nd infusion Management: CRS management (Appendix F) *Patients requiring a delay of > 7 days will not receive the 2nd dose of modified CAR-20/19-T cells   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
figure 1 study schema
dose modifications and dosing delay

Each patient receiving CAR-20/19-T cells will be evaluable for safety. The safety parameters include all laboratory tests and hematological abnormalities, physical findings, and spontaneous reports of adverse events reported to the investigator by patients. Each patient will be assessed periodically for any toxicity development as per the schedule of procedures (Table 1). Toxicity will be assessed per the NCI CTCAE v5.0 except for CRS which will be graded as previously defined.  
For expected cytopenias patients will be transfused platelets and red blood cells as per
figure 1 study schema
appendix g.

We will monitor specifically for the development of CRS, a known complication that can occur with the treatment of CAR modified T cells. Signs/symptoms related to CRS were listed in section 1.7 (**Figure 8**). Grading of CRS will be by the revised recommended grading system for CRS found in **Appendix F**. Any patient who develops fever as an outpatient will be admitted for work-up and evaluation of CRS. Detailed algorithm for CAR-T cell associated CRS or neurotoxicity based on current recommendations can be found in **Appendix F.** The current recommendations for CRS management include Tocilizumab and/or steroids as first-line therapies. Tocilizumab is a humanized, IgG1к antihuman IL-6R monoclonal antibody approved for the treatment of several rheumatological conditions. Tocilizumab prevents IL-6 binding to both cell associated and soluble IL-6Rs inhibiting signaling and has led to clinical improvement in patients with life-threatening CRS within 24 hours of administration.[1, 53] For patients failing to respond after two doses of tocilizumab, steroids will be second line for CRS. Further management beyond second line therapy will depend upon the judgment of the clinician and may include additional immunosuppression such as Siltuximab or other agents that can inhibit T cell proliferation/function.
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7.3 other toxicities

Other toxicities include those associated with lymphodepleting chemotherapy regimen which includes fludarabine with cyclophosphamide.  
| Hematologic Toxicities   | Grade 3 or 4 anemia, neutropenia, and thrombocytopenia are  potential toxicities associated with lymphodepleting chemotherapy.   |
|--------------------------|----------------------------------------------------------------------------------------------------------------------------------|
| Infection                | Neutropenic fever is a potential toxicity associated cytotoxic  chemotherapy.                                                    |
| Tumor Lysis Syndrome     | Can occur as a result of tumor cell death with treatment with  cytotoxic chemotherapy.                                           |
| Organ toxicity           | Can be pulmonary, renal, liver, or cardiac toxicity related to  chemotherapy administration                                      |
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8 adverse events definitions and

The investigator and his or her team will follow the Medical College of Wisconsin policies related to adverse event reporting. This information may be found on the Human Research Protection Program website. (http://www.mcw.edu/hrpp/InvestigatorsandStudyStaff.htm) Serious AE (SAE) means any untoward medical occurrence that at any dose:
- **Death.** Results in death.  
- **Life threatening**. Is life threatening (refers to an AE in which the patient was at risk of death at the time of the event. It does not refer to an event which hypothetically might have caused death if it were more severe).  
- **Hospitalization**. Requires inpatient hospitalization or prolongation of an existing hospitalization (see clarification in the paragraph below on planned hospitalizations).  
- **Disability/incapacity**. Results in persistent or significant disability or incapacity.  
(Disability is defined as a substantial disruption of a person's ability to conduct normal life functions).  
- **Medically important event**. This refers to an AE that may not result in death, be immediately life threatening, or require hospitalization, but may be considered serious when, based on appropriate medical judgment, may jeopardize the patient, require medical or surgical intervention to prevent 1 of the outcomes listed above, or involves suspected transmission via a medicinal product of an infectious agent. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization; any organism, virus, or infectious particle (e.g., prion protein transmitting Transmissible Spongiform Encephalopathy), pathogenic or nonpathogenic, is considered an infectious agent.  
Clarification should be made between a serious AE (SAE) and an AE that is considered severe in intensity (Grade 3 or 4), because the terms serious and severe are NOT synonymous. The general term severe is often used to describe the intensity (severity) of a specific event; the event itself, however, may be of relatively minor medical significance (such as a Grade 3 headache). This is NOT the same as serious, which is based on patient/event outcome or action criteria described above, and is usually associated with events that pose a threat to a patient's life or ability to function. A severe AE (Grade 3 or 4) does not necessarily need to be considered serious. Seriousness (not intensity) serves as a guide for defining regulatory reporting obligations. Planned hospitalizations (e.g. admission for CAR-T infusion) will not be considered an adverse event.
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8.2 unanticipated problem involving risk

The investigator and his or her team will follow the Medical College of Wisconsin policies related to unanticipated problems involving risks to subjects or others. This information may be found on the Human Research Protection Program website
(http://www.mcw.edu/hrpp/InvestigatorsandStudyStaff.htm)
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8.3 ae attribution and grading

Will be performed using the NCI CTCAE Version 5.0 except for CRS which will be graded as described in Appendix F per clinical guidelines.
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adverse event attribution

Attribution is an assessment of the relationship between the AE and the medical intervention.
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relationship assessment in-depth definitions

For all collected AEs, the clinician who examines and evaluates the subject will determine the adverse event's causality based on temporal relationship and his/her clinical judgment. The degree of certainty about causality will be graded using the categories below:
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible contributing factors can be ruled out. The clinical event, including an abnormal laboratory test result, occurs in a plausible time relationship to CAR-20/19-T cell administration and cannot be explained by concurrent disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be clinically plausible. The event must be pharmacologically or phenomenologically definitive, with use of a satisfactory rechallenge procedure if necessary.  
Probably Related: There is evidence to suggest a causal relationship, and the influence of other factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within a reasonable time sequence to CAR-20/19-T cell administration, is unlikely to be attributed to concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on withdrawal (dechallenge). Rechallenge information is not required to fulfill this definition.  
Possibly Related: There is some evidence to suggest a causal relationship (e.g., the event occurred within a reasonable time after administration of the trial medication). However, the influence of other factors may have contributed to the event (e.g., the subject's clinical condition, other concomitant events). Although an adverse event from CAR-20/19-T cell infusion may rate only as "possibly related" soon after discovery, it can be flagged as requiring more information and later be upgraded to "probably related" or "definitely related", as appropriate.  
Unlikely: A clinical event, including an abnormal laboratory test result, whose temporal relationship to CAR-20/19-T cell administration makes a causal relationship improbable (e.g., the event did not occur within a reasonable time after administration of the trial medication) and in which other drugs or chemicals or underlying disease provides plausible explanations (e.g., the subject's clinical condition, other concomitant treatments).  
Unrelated: The AE is completely independent of CAR-20/19-T cell administration, and/or evidence exists that the event is definitely related to another etiology. There must be an alternative, definitive etiology documented by the clinician.
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known aes list

Potential risks associated with this treatment include symptoms/complications associated with CRS and neurotoxicity. Section 1.7 lists the known symptoms/complications associated with CRS and other known risks of CAR-T cell administration.
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8.4 monitoring and recording an

Definition. Any clinically relevant deterioration in laboratory assessments or other clinical finding is considered an AE.  
Reporting source. AEs may be spontaneously reported by the patient and/or in response to an open question from study personnel or revealed by observation, physical examination or other diagnostic procedures.  
Prior to the trial. Planned hospital admissions or surgical procedures for an illness or disease that existed before the patient was enrolled in the trial are not to be considered AEs unless the condition deteriorated in an unexpected manner during the trial (e.g., surgery was performed earlier or later than planned).  
Pretreatment events following signed informed consent. For serious pretreatment events, the investigator must determine both the intensity of the event and the relationship of the event to study procedures.  
Treatment events. For serious AEs, the investigator must determine both the intensity of the event and the relationship of the event to study drug administration. Not serious AEs. For non-serious AEs, the investigator must determine both the intensity of the event and the relationship of the event to study drug administration.
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follow-up of adverse events

All adverse events will be followed with appropriate medical management 30 days following the last dose of the study drug or treatment or until they are resolved, if they are related to the study treatment.
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8.5 procedure for reporting drug

If a woman becomes pregnant, or suspects that she is pregnant, while participating in this study, she must inform the investigator immediately. The sponsor-investigator must notify the DSMC and IRB. The pregnancy must be followed for the final pregnancy outcome. As this study utilizes an investigational agent under IND with the FDA, this will be reported to the FDA. If a female partner of a male patient becomes pregnant during the male patient's participation in this study, the sponsor-investigator must also immediately notify the DSMC by email. Every effort should be made to follow the pregnancy for the final pregnancy outcome.
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8.6 subject complaints

If a complaint is received by anyone on the study staff, it will be discussed with the study staff and will be addressed on a case-by-case basis. The PI will be notified of any complaints. Complaints will be reported to the IRB if indicated. If the subject has questions about his or her rights as a study subject, wants to report any problems or complaints, obtain information about the study or offer input, the subject can call the Medical College of Wisconsin/Froedtert Hospital research subject advocate at 414-9558844. This information is provided to the subject in their consent.  
A product complaint is a verbal, written or electronic expression that implies dissatisfaction regarding the identity, strength, purity, quality or stability of a drug product. Individuals who identify a potential product complaint situation should immediately contact the sponsor and report the event. Whenever possible, the associated product should be maintained in accordance with the label instructions pending further guidance from a sponsor representative. Product complaints in and of themselves are not Reportable Events. If a product complaint results in an SAE, an SAE form should be completed.
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8.7 routine reporting procedures for

Expedited Reporting Procedures for SAEs, UPIRSOs and DLTs. Since this is an investigator-initiated study, the principal investigator, also referred to as the sponsor-investigator, is responsible for reporting serious adverse events (SAEs) to any regulatory agency and to the sponsor- investigator's IRB. Regardless of expectedness or causality, all SAEs (including serious pretreatment events) must also be reported to the DSMC as soon as possible, but no later than five calendar days of the sponsor-investigator's observation or awareness of the event.  
Signs or symptoms reported as adverse events will be graded and recorded by the investigator, according to the CTCAE. When possible, signs and symptoms indicating a common underlying pathology should be noted as one comprehensive event. The investigator will assess all adverse events and determine reporting requirements to the MCWCC Data and Safety Monitoring Committee (DSMC) and MCW's Institutional Review Board, and, when the study is conducted under an Investigational New Drug Application (IND), to the Food and Drug Administration (FDA), if it meets the FDA reporting criteria. The investigator will report SAEs to any regulatory agency and to the sponsor- investigator's IRB. All adverse events, whether or not unexpected, and whether or not considered to be associated with the use of the study drug, will be entered into OnCore®.
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reporting to the data and

Regardless of expectedness or causality, all SAEs (including serious pretreatment events) must also be reported to the DSMC as soon as possible, but no later than **five calendar days** of the sponsor-investigator's observation or awareness of the event.  
Report Method: The investigator will use email to report SAEs to the DSMC. The SAE report must include event term(s), serious criteria, and the sponsor-investigator's or sub-investigator's determination of both the intensity of the event(s) and the relationship of the event(s) to study drug administration. Intensity for each SAE, including any lab abnormalities, will be determined by using the NCI CTCAE as a guideline whenever possible.  
The criteria are available online at http://ctep.cancer.gov/reporting/ctc.html.
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reporting to mcw committee institutional

The principal investigator must report unanticipated problems involving risks to subjects or others (UPIRSOs) to the MCW IRB within five business days of his/her awareness of the event.  
[Guidance on Adverse Event Reporting to the IRB is available online at MCW IRB Policies and Procedures.] (http://www.mcw.edu/hrpp/policiesprocedures.htm)
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expedited reporting to the food

As this study is being conducted under an IND, the sponsor-investigator is responsible for determining whether the suspected adverse reaction meets the criteria for expedited reporting in accordance with Federal Regulations (21 CFR §312.32).  
The investigator will report in an IND safety report any suspected adverse reaction that is both serious and unexpected. In addition, per FDA recommendations, the sponsor-investigator will report all CRS ≥Grade 3 and Neurotoxicity ≥Grade 3 that occurs as a result of CAR-20/19-T cell infusion. For other events, the sponsor-investigator needs to ensure that the event meets all three definitions: *suspected adverse reaction, unexpected, and serious* If the adverse event does not meet one of the above definitions, it should not be submitted as an expedited IND safety report. The timeline for submitting an IND safety report to FDA is no later than **15 calendar days** after the investigator determines that the suspected adverse reaction qualifies for reporting (21 CFR 312.32(c)(1)). Any unexpected fatal or life-threatening suspected adverse reaction will be reported to FDA no later than **seven calendar days** after the Investigator's initial receipt of the information (21 CFR
312.32(c)(2)).  
Any relevant additional information that pertains to a previously submitted IND safety report will be submitted to FDA as a Follow-up IND Safety Report without delay, as soon as the information is available (21 CFR 312.32(d)(2)).
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suggested reporting form

US FDA MedWatch 3500A:
http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.htm Any other form deemed appropriate by the sponsor-investigators
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9 pharmaceutical information 9.1.1 car-20/19-t

Product Description Each CAR-20/19-T cell line is a unique product prepared from patient peripheral blood CD4+ and CD8+ T cells. The cells have been engineered to express two different extracellular single chain antibodies (scFv) recognizing CD20 and CD19. The antibodies are linked to tandem signaling domains comprised of the CD3ζ signaling module linked to the 4-1BB costimulatory domain (**Figure 3**). The CAR-20/19-T cells if given fresh without cryopreservation will be in an infusion medium of an isotonic electrolyte solution (Plasma-Lyte® A or Normosol® R) containing 2.5% HSA. If needed, the CAR-T cells will be cryopreserved in infusible grade cryomedium at two different cell numbers. For cryopreserved cells the infusion medium will consist of 10% dimethylsulfoxide, 4% human serum albumin, in 85% Normosol R.
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mechanism of action

When the antibody chains on the CAR-20/19-T cells bind to their ligands on the surface of B cells the T cells are triggered to kill the cells to which they bind. A given T cell can bind and kill multiple target cells.
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metabolism

The CAR-20/19-T cells will be activated by ligand engagement and are expected to expand in vivo. Transduced cells are expected to persist for a period of months to years.
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side effects

Side effects are thoroughly detailed in section 1.7. Briefly major toxicities include:  
- B cell ablation- Since CD19 and CD20 are also expressed on healthy B cells, the CAR20/19-T cells are expected to eliminate these cells from the circulation as long as the cells persist. The loss of B cells could result in an increased risk of infection.  
- Allergic Reaction- The scFv on the T cell surface may invoke an immune reaction that might eliminate the CAR-20/19-T cells and secondarily cause an autoimmune response, though such responses have not been reported in clinical trials to date.  
- Transformation- The genetic modification of the CAR-20/19-T cells could result in transformation. To date transformations of CAR-T cells have not been reported
- Tumor lysis syndrome (TLS) - Patients with a high tumor burden may experience a reaction due to lysis of the tumor cells either from the lymphodepleting preparative regimen or from the CAR-20/19-T cells. Such reactions have been reported and can be severe. With careful monitoring, appropriate therapies are available that can mitigate this reaction.  
- Cytokine Release Syndrome (CRS)- In vivo expansion of the infused CAR-20/19-T cells may be associated with a release of inflammatory cytokines. Such responses have been reported, some have been severe. Close monitoring and appropriate interventions are available that can mitigate this reaction.  
- Neurotoxicity- CAR-T therapy has led to some patients having unpredictable neurotoxicity in the form of seizures, aphasias, and in rare cases life threatening cerebral edema. Clinical vigilance and early intervention can help prevent and/or treat this complication.  
- Unknown- Given this first-in-human use of CAR-20/19-T cells unknown side effects may occur either associated with the infusion or long term. Monitoring for such effects is described in this protocol
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9.1.2 solution preparation

The CAR-20/19-T cells will be administered either fresh following harvest or within 6 weeks of cryopreservation in the appropriate doses for infusion. Cryopreserved cells will be thawed within the LPL prior to administration. The CAR-T cells will be transported at 1-10oC to the infusion site. If the dose level changes from the time of product cryopreservation to time of infusion, the volume of product infused will be adjusted accordingly.
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9.1.3 investigational agent administration

The cells will be administered from 1-7 days after completion of chemotherapy to deplete lymphocytes. The patient will be premedicated. Vital signs will be monitored 15 minutes prior to infusion and every 15 minutes after infusion for the first hour. Monitoring will be continued until all signs are stable. The cells will be administered over a period of 5-15 minutes by IV injection.  
See Section 6 for further details.
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9.1.4 storage requirements

For patients requiring cryopreservation, the cells will be stored cryopreserved in the LPL until the time of infusion. Cryopreservation storage is in vapor phase liquid nitrogen at temperatures
<-150oC in devices that are continuously monitored for temperature according to standard laboratory practices.  
Fresh cells will be stored at 1-10oC in a monitored refrigerator and will be given within 48 hours of harvest or cryopreserved accordingly.
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9.1.5 stability

Fresh cells have been demonstrated to be stable in infusion medium up to 48 hours after harvest when stored at a concentration <20 x 106 per mL at 1-10oC. Longer term phenotypic and functional stability data up to 1 year is shown in the below table and Figure 11. Based on the 1-year data, the product is likely stable indefinitely while in the cryopreserved state. Once thawed, the cell infusion must start within 45 minutes.  
| CAR-T Cell Phenotype Pre and Post Cryopreservation   |                        |        |        |        |       |       |
|------------------------------------------------------|------------------------|--------|--------|--------|-------|-------|
| Subjects                                             | Time from Cryo to Thaw | D3     | CD4    | CD8    | NK    | NKT   |
| SHAH-002                                             | Fresh product          | 9.18%  | 57.60% | 48.34% | 0.50% | 5.60% |
| SHAH-002                                             | 1 month                | 97.03% | 52.94% | 46.80% | 0.20% | 0.60% |
| SHAH-002                                             | 3 month                | 9.18%  | 48.27% | 51.41% | 0.30% | 2.30% |
| SHAH-002                                             | 1 Year                 | 9.63%  | 53.90% | 48.00% | 0.10% | 2.50% |
| SHAH 003                                             | Fresh product          | 9.30%  | 62.11% | 39.18% | 0.00% | 0.00% |
| SHAH 003                                             | 9.38%                  | 60.14% | 40.15% | 0.00%  | 0.50% |       |
| 2 weeks                                              |                        |        |        |        |       |       |
| SHAH 003                                             | 1 month                | 98.93% | 59.97% | 41.25% | 0.10% | 0.60% |
| SHAH 003                                             | 6 month                | 9.75%  | 53.79% | 47.62% | 0.00% | 0.70% |
| SHAH 003                                             | 1 Year                 | 9.93%  | 61.22% | 40.75% | 0.00% | 0.40% |
| SHAH 004                                             | Fresh product          | 9.83%  | 64.34% | 42.26% | 0.00% | 0.50% |
| SHAH 004                                             | 6 month                | 9.85%  | 58.64% | 45.13% | 0.00% | 0.20% |
| SHAH 004                                             | 1 Year                 | 9.85%  | 53.32% | 50.72% | 0.00% | 0.60% |
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phenotypic stability of car-20/19-t cell

A.  Percent CAR+ T Cells B. CD19+ Target Cells C. CD20+ Target Cells  
  
Protein
Figure 11: Phenotypic and Functional Stability of CAR-20/19-T Cell Products. CAR-20/19-
T cell products generated from Subjects 002-004 in the Phase I trial were cryopreserved, thawed at the indicated time points to assess (A) Protein L positivity to assess surface CAR expression, (B) cytotoxicity versus CD19+ target cells, and (C) cytotoxicity versus CD20+ target cells. Cell cytotoxicity was conducted at an effector:target (E:T) cell ratio of 50:1 ( note: the E:T ratio was not adjusted based on the percentage of CAR-T cell present in the product; it was based on total T cells).
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9.1.7 nursing implications

Premedication and patient monitoring before, during, and after each split dose infusion is required. See Section 6 for details
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9.1.8 handling

The required dose of CAR-20/19-T cells are transported at 1-10oC. The cells are infused at room temperature over a 5-15-minute period by IV injection. For cryopreserved cells, infusion should start no longer than 45 minutes after thawing.
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9.1.9 availability

Each lot of CAR-20/19-T cells is patient-specific. The cell lines are produced on site by the MCW cell processing laboratories.
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9.1.10 agent ordering

CAR-20/19-T cells are produced at MCW and are a patient-specific material. A physician order for infusion specifying the dose of transduced cells per kg to be infused is required.
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9.1.11 agent accountability

CAR-20/19-T cells are identified by a donor identification number (DIN) that is used to track the product at collection to the product at disposition (infused or discarded) and to trace the product at infusion to the donation. CAR-20/19-T cells are for autologous use only and are labeled accordingly.
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9.1.12 agent destruction and return

Once issued from the LPL, the CAR-20/19-T cell line cannot be returned for reissue. Aliquots of the lines must be maintained for potential further analysis; however, the cells are for autologous use only and may not be released to another patient.
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9.2.1 tocilizumab product description

Tocilizumab is a monoclonal antibody and immunosuppressant; specifically, tocilizumab is an interleukin-6 (IL-6) receptor antagonist. IL-6 is a pleiotropic pro-inflammatory cytokine and is produced by various cells including T- and B cells, lymphocytes, monocytes and fibroblasts. This agent will be part of the first line treatment for patients with CRS requiring more than supportive measures for medical management (Appendix F for CRS management guidelines).
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mechanism of action

Tocilizumab binds IL-6 receptors (sIL-6R and mIL-6R) and inhibits IL-6-mediated signaling through these receptors. IL-6 has been shown to be involved in T cell activation, induction of immunoglobulin secretion, initiation of hepatic acute phase protein synthesis, and stimulation of hematopoietic precursor cell proliferation and differentiation.
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contraindications

Administration of live vaccines is contraindicated during treatment with tocilizumab. Use is contraindicated in patients with known hypersensitivity to tocilizumab.
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drug interactions

Use of tocilizumab may cause increased metabolism of medications that are CYP450 substrates including CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Caution should be used in medications that are CYP450 substrates including simvastatin, dextromethorphan and omeprazole, and medications with a narrow therapeutic index.
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side effects

- Myelosuppression: Tocilizumab may lead to neutropenia and thrombocytopenia.  
- Hepatic enzyme and lipid parameter alterations: Tocilizumab may lead to an elevation in liver enzymes and an increase in lipid parameters including: total cholesterol, triglycerides, LDL cholesterol, and HDL cholesterol.  
- Immunosuppression: Tocilizumab may lower patient's resistance to infections.  
- Gastrointestinal perforation: Events have occurred in clinical trials, caution in patients at increased risk of developing gastrointestinal perforation
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9.2.2 preparation

Tocilizumab should be diluted in 100 mL of 0.9% sodium chloride. Prior to addition of tocilizumab, withdraw a volume of 0.9% sodium chloride from the infusion bag equal to the volume of tocilizumab required (total volume of final preparation should be 100 mL). Gently mix the infusion to avoid foaming
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9.2.3 tocilizumab administration

Tocilizumab should be administered over 60 minutes as an intravenous infusion. The infusion should have a dedicated line and not be infused concomitantly with other drugs.
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9.2.4 storage

Vials must be refrigerated at 2ºC to 8ºC (36°F to 46°F). Do not freeze. Vials should be protected from light and stored in the original package until time of use. Prepared parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration. Do not use the product if visibly opaque particles, discoloration, or other foreign particles are observed in the solution.
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9.2.5 stability

Prepared solutions may be stored at 2ºC to 8ºC (36°F to 46°F) or room temperature for up to 24 hours.
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9.2.7 availability

Tocilizumab is available in single dose vials (20 mg per mL) of 80 mg per 4 mL, 200 mg per 10 mL, and 400 mg per 20 mL.
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10 reporting and documenting response

Evaluable for toxicity
- All patients will be evaluable for toxicity from the time of their first study drug treatment. Evaluable for objective response - Only those patients who have received CAR-20/19-T cells will be re-evaluated and considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the first disease assessment at Day +28 will also be considered evaluable.)
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10.2 response criteria

Response assessment will vary by disease subtype and specific criteria are listed below for CLL/SLL and NHL.
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10.3 chronic lymphocytic leukemia response

Response criteria as per 2008 guidelines for diagnosis and treatment of CLL/SLL [61]  
Complete Response (CR)
1. Peripheral blood lymphocytes (evaluated by blood and differential count) below 4.0 x 109/L (4000/µL)
2. Absence of significant lymphadenopathy (e.g., lymph nodes >1.5 cm in diameter) by physical examination and CT scan 3. No hepatomegaly or splenomegaly by physical examination or CT imaging 4. Absence of constitutional symptoms 5. ANC >1500/µL, Hemoglobin >11.0 g/dL, Platelets >100,000/ µl 6. Bone Marrow Biopsy confirmation
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complete response with incomplete marrow

1. Fulfill all criteria for a CR but have persistent anemia, thrombocytopenia, or neutropenia felt to be unrelated to CLL but related to drug toxicity.
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partial remission (pr)

1. A decrease in the number of blood lymphocytes by 50% or more from value before therapy 2. Reduction in lymphadenopathy by CT scans as defined by the following a. A decrease in lymph node (LN) size by 50% or more either in the sum products of up to 6 lymph nodes, or in the largest diameter of the enlarged node(s) detected prior to therapy b. No increase in any LN and no new enlarged LN. In small LN (<2 cm), an increase of less than 25% is not considered to be significant.  
3. A reduction in the pretreatment enlargement of the spleen or liver by 50% or more as detected by CT scan 4. Blood count should show one of the following results a. Neutrophils >1500/µl without exogenous growth factors b. Platelet count >100,000/µl or 50% improvement over baseline without need for exogenous growth factors c. Hemoglobin >11.0g/dL or 50% improvement from baseline without requiring RBC
transfusions or exogenous erythropoietin.  
Progressive Disease 1. Lymphadenopathy a. Appearance of any new lesion, such as enlarged lymph nodes (>1.5 cm),
splenomegaly, hepatomegaly, or other organ infiltrates.  
b. An increase by 50% or more in greatest determined diameter of any previous site.  
2. An increase in the previously noted enlargement of the liver or spleen by 50% or more or the de novo appearance of hepatomegaly or splenomegaly.  
3. An increase in the number of blood lymphocytes by 50%or more with at least 5000 B
lymphocytes per microliter.  
4. Transformation to a more aggressive histology (e.g., Richter syndrome). Whenever possible, this diagnosis should be established by lymph node biopsy.  
5. Occurrence of cytopenia (neutropenia, anemia, or thrombocytopenia) attributable to CLL
Stable Disease 1. Patients who have not achieved a CR or a PR, and who have not exhibited progressive disease, will be considered to have stable disease (which is equivalent to a nonresponse).
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10.4 non-hodgkin's lymphoma response criteria

2014 Lugano Classification will be utilized for response assessment in patients with NHL. [62]
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complete response (cr) pet/ct based

o Complete metabolic response: Score 1, 2, 3 with or without a residual mass on the 5 point Deauville Scale
(**Figure 12**)
o No evidence of FDG-avid disease in marrow CT-Based Response o Target nodes/nodal masses must regress to ≤1.5 cm in longest transverse diameter
(LDi)
o No extralymphatic sites of disease o Negative BM biopsy if positive at baseline o Regression of organ enlargement to normal
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figure 12 5 point deauville

o Score 4 or 5 on Deauville scale with reduced uptake compared with baseline and residual masses of any size CT-Based Response o ≥50% decrease sum of the product of the perpendicular diameters (SPD) of up to 6 dominant measurable nodes and extranodal sites; no criteria for progressive disease are met o When a lesion is too small to measure on CT, assign 5 mm x 5 mm as the default value. When no longer visible, 0 x 0 mm o For a node >5 mm x 5 mm, but smaller than normal, use actual measurement for calculation o If enlarged, spleen must have regressed by >50% in length
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stable disease (sd)

PET/CT Based Response o Score 4 or 5 on Deauville scale with no significant change in FDG uptake from the baseline scan
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ct-based response

o <50% decrease from baseline SPD of up to 6 dominant measurable nodes and extranodal sites, no criteria for progressive disease are met.  
o No increase in non-measured lesions consistent with progression o No increased in organ enlargement consistent with progression.
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progressive disease (pd) pet/ct based

o Score 4 or 5 on Deauville scale with increase in intensity of uptake from baseline o New FDG-avid foci consistent with lymphoma o New or recurrent FDG-avid foci in bone marrow
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ct-based response

o An individual node/lesion must be abnormal with longest transverse diameter >1.5 cm and have an increase by ≥50% from cross product of the LDi and perpendicular diameter and have an increase in the LDi or shortest axis perpendicular (SDi) from nadir as follows:
o 0.5 cm for lesions ≤2 cm o 1.0 cm for Lesions >2 cm o In the setting of splenomegaly, the splenic length must increase by >50% of the extent of its prior increase beyond baseline (eg: a 15-cm spleen must increase to >16 cm). If no prior splenomegaly, must increase by at least 2 cm from baseline o New or recurrent splenomegaly o New or clear progression of pre-existing non-measured lesions o Re-growth of previously resolved lesions o A new node >1.5 cm in any axis o A new extra nodal site > 1.0 cm in any axis; if <1.0 cm in any axis is presence must be unequivocal and must be attributable to lymphoma o Assessable disease of any size unequivocally attributable to lymphoma
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10.5 response definitions evaluation of

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria. The first disease assessment will occur at 4 weeks' post treatment (Day+28). Response assessment will continue up to 2 years or until progression/death.
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duration of response duration of

The overall response duration is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started).  
The overall CR duration is measured from the time measurement criteria are first met for CR until the first date that recurrent disease is objectively documented.
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duration of stable disease

Stable disease is measured from the start of the treatment until the criteria progression are met, taking as reference the smallest measurements recorded since the treatment started.
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progression-free survival

Progression-free survival (PFS) is defined as the time duration from treatment start to progression time.
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10.6 evaluation of safety

Analyses will be performed for all patients having received at least one dose of study drug. The study will use the CTCAE v5.0 for reporting of non-hematologic adverse events and modified criteria for hematologic adverse events.
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11 correlative studies/special studies 11.1

Diversity of the T cell repertoire (as measured by T cell receptor sequencing) has been associated with effective T cell immunity. Conversely, low levels of T cell diversity have been correlated with aging, infection, and the development of cancer. The relation between the diversity of the T cells used for CAR-T therapy and outcomes are not understood. Neither is the effect of generating CAR-T on repertoire diversity understood. Both could be important parameters in therapy outcomes.  
We will measure the diversity of the circulating T cell repertoire at three time points during the study to examine: 1) the effect of generating the CAR-T cells and 2) any possible effect of infusion of the CAR-T product on the overall circulating repertoire of subject. The initial sample of 107 mononuclear cells will be obtained at time of apheresis and the T cell repertoire diversity will act as the benchmark for the two comparative analyses. The second sample will consist of the same number of cells taken from the final CAR-T product prior to infusion into the subject.  
The final sample will consist of 10ml of blood and will be obtained as part of the 28-day disease assessment.  
- Sample 1: Will be obtained from the apheresis product collected at Day -15
- Sample 2: From final CAR-T cell product prior to patient infusion - Sample 3: Peripheral blood sample (10 ml) on Day +7 - Sample 4: Peripheral blood sample (10 ml) on Day +28 (+/- 3 days)
The samples will be labeled with a unique patient identifier and sent to BloodCenter of Wisconsin (BCW) for T cell repertoire analysis. At BCW the de-identified samples will be subject to cell separation into CD4 and CD8 T cells, cDNA tagged with unique barcodes will be prepared and subject to BV gene-specific amplification and single cell RNA sequencing (scRNA-Seq). The amplicons will be pooled and subject to NGS sequencing on the Illumina platform. The sequence data will be clustered on the basis of unique CDR3 sequence (clonotype), whole transcriptome and the number of unique cDNA-defining barcodes counted to quantify the clonotype. Diversity and complexity measures will be derived, the latter involving not just the number of unique species but their frequency distribution. A similar analysis will be performed on the basis of the amino acids encoded by the CDR3 sequence (motif analysis). As needed the depth of sequencing can be modulated to bring up the resolution level.  
The repertoire diversity analyses and scRNA-Seq will describe the initial diversity of the repertoire and show whether the CliniMACS Prodigy-derived CAR-T cells have undergone major shifts in the T cell population being infused into the patient. Both results will be most informative when compared to other clinical parameters and outcomes. Comparison of the initial circulating repertoire diversity with that of the circulating repertoire after infusion of the CAR-T product will show if there have been significant changes in the repertoire and differentiation states due to the functioning of the CAR-T cells, and comparison of the data with clinical outcomes should be informative. The risk associated with these analyses is minimal. The lab at BCW will be working with de-identified samples. The investigators at BCW are experts in this area of analysis. The information gained will be useful for the overall interpretation of outcomes.
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11.2 circulating tryptophan metabolites and

Cytokine release syndrome (CRS) is a systemic inflammatory reaction that is observed in a high proportion of patients receiving CAR-T-based therapies. CRS is driven by an increase in the release of proinflammatory cytokines that are released after the activation of monocytes, macrophages and some lymphocyte populations. In addition to CRS, and perhaps as a consequence, patients receiving CAR-T therapies also experience CNS toxicity, including tremor, seizures and life-threatening cerebral edema. Although the pathophysiology of these neurological effects is not clear, inflammatory cytokines are thought to be involved. Human studies, primarily carried out in patients with major depressive illness, indicate that the kynurenine pathway is a critical link between inflammation and damage to the brain. The kynurenine pathway is activated by inflammatory cytokines (predominantly interferon gamma) released in the brain by activated T cells and leads to the production of kynurenine from tryptophan as a result of up-regulation of the enzyme indoleamine 2,3-dioxygenase (IDO) by microglia or brain-resident macrophages. Kynurenine is further metabolized to neurotoxic metabolites 3-hydroxykynurenine (3-HK) and quinolinic acid (QA) and to the neuroprotective metabolite, kynurenic acid (KA). The ratios of serum KA/3-HK and/or KA/QA have been used previously as indices of the role of this inflammatory pathway on brain function. Low ratios are seen in patients with mood disorders, for example, compared to controls. Further, we have identified a gene expression profile of 53-genes termed the "conserved transcriptional response to adversity" (CTRA), a gene expression pattern associated with exposure to chronic stressors and whose largest gene subset is comprised of proinflammatory genes. This CTRA gene expression has been associated with worse outcomes following HCT. We hypothesize that neuroinflammation is responsible for the adverse effect of CAR-T cell therapy on the brain and that the serum KA/3-HK and KA/QA ratios as well as CTRA profiles will serve as biomarkers for neuroinflammation, and thus, as biomarkers for the risk of CNS toxicity. We will measure the serum concentrations of tryptophan, kynurenine, 3-HK, QA, KA, and gene expression patterns (CTRA) at 4 time points during the study to examine: (1) the trajectory in the concentration of each metabolite and gene expression in the month following CAR-T cell therapy; and (2) the correlation between the KA/3-HK and KA/QA ratios as well as gene expression and the occurrence of CNS toxicity. A baseline sample will be assessed on the day of apheresis and will serve as a benchmark for the trajectory study. Two additional blood samples will be analyzed during the first month after treatment (14 and 28 days) and one sample will be analyzed after three months (day 90) to assess recovery. The samples will be collected along with other blood samples at these time points in the study. The samples will be collected via standard venipuncture in serum separating tubes (metabolites) and PaxGene RNA tubes (gene expression) and will be collected using standard protocols and stored at -80 degrees in cryotubes labeled with unique patient identifiers until analysis. The samples will be sent to the laboratory of Dr. Cecilia Hillard in the Neuroscience Research Center. The serum metabolite tubes will be processed for the concentrations of 5 analytes: tryptophan, kynurenine, 3-HK, QA and KA using high performance liquid chromatography with tandem mass spectrometry. The extraction and derivatization of the samples will be carried out in the Hillard laboratory and the mass spectroscopy will be performed in the Department of Pharmacology at the Medical College of Wisconsin. The PaxGene RNA tubes will be stored until they can be shipped in batch to the UCLA Social Genomics Core (Directed by Dr. Steve Cole, Professor, Hematology/Oncology, UCLA), who will conduct all gene expression profiling on obtained blood samples by using Illumina HT-12 human gene expression bead arrays. Total RNA will be extracted from the whole blood samples stored at MCW, subjected to quality assurance assays to test suitable mass (by spectroscopy) and integrity (by Agilent Bioanalyzer RNA Integrity Score) for analysis, and subjected to microarray target synthesis and hybridization in collaboration with the UCLA Neuroscience Genomics Core Laboratory using standard Illumina assay equipment and protocols. The output of these analyses is quantification of whole genome RNA production. The laboratory staff will work with de-identified samples and the data will be provided to the study PI for correlation with CNS toxicity in the same patients. The information gathered from this study will provide important mechanistic information regarding the relationships among the tryptophan-kynurenine pathway, proinflammatory gene expression, and the occurrence of CNS toxicity, and could elucidate these metabolites and gene expression clusters as potential biomarkers for the occurrence of CNS toxicity in patients undergoing CAR-T therapy.
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11.3 patient-reported outcome (pro) measures

Related to or as a consequence of CNS and its neurotoxicities, including neuroinflammation, patients receiving CAR-T therapies may be at risk for diminished quality of life and patientreported outcomes (PROs), including the development of depression, anxiety, fatigue, sleep disturbances, and pain. There are several mechanistic modalities by which engineered autologous CAR-T cells may influence and/or be affected by PRO measures. First, immune activation, inflammation, and cytokine activation - as occurs with CRS - are known to affect cognitive and/or emotional functioning,[63] including among cancer patients.  
[64] Second, while patient-reported symptomatology may be less salient during the initial acute period of potentially serious illness, as can occur with CRS [1, 53, 54], the acute and likely subsequent inflammation may play a key role in the development of depression and other psychopathologic symptoms; it is unknown what longer-term QOL sequelae may be present after an initial severe (and potentially ongoing) inflammatory insult. Prior research indicates that inflammation itself is involved in both the genesis and foreshadowing of future depressive or psychotic episodes.[65] Third, altered central nervous system (CNS) functioning and subsequent emotional/cognitive responses influence immunity and T cell function among cancer patients.[66]Further, these adverse responses are predictive of compromised immune reconstitution[67] and adverse outcomes following HCT.[68-70]Therefore, it is critical to assess whether or how CNS and psychological functioning will impact the efficacy of this new and likely increasingly used cancer treatment modality. As infection remains a significant risk of this therapy, it will be important to understand and optimize any factors that could contribute to increased infectious susceptibility, including neuroimmunologic influence.  
Finally, it is unknown what longer-term (1-2 years and beyond) effects such enhanced immune activation will have. Given the aforementioned immune/CNS effects and increasing emphasis on QOL and PROs as mainstay parameters in assessing cancer treatment efficacy,[71, 72] it is important to ascertain late effect PROs.  
We will measure the following PROs (see **Appendix I** for questionnaires) during the ancillary study at the same time points as the biomarkers described in section 11.2 (at apheresis, D+14, D+28, D+90), as well as at the additional time points of 6 months, 1 year, and 2 years:
Depression: Depression will be assessed through the General Depression subscale of the Inventory of Depression and Anxiety Symptoms (IDAS) and includes 20 questions with a scoring range of 20-100 (mean in community dwelling adult of 44.99 and standard deviation of 14.75).[73] Anxiety: Anxiety will be assessed using two subscale items of the IDAS including panic (health population mean = 12.58, SD = 5.26) and traumatic intrusions (healthy population mean = 7.60, SD = 4.20)[73]
Fatigue: The Fatigue Severity Index (FSI) will be utilized to assess fatigue; a score of 3 or greater on items assessing fatigue in the past week (average of items 1-3; FSI
Composite) indicates clinically meaningful fatigue.[74] The FSI can also be evaluated using the average rating of the degree to which fatigue interfered with some general activities (0-10; FSI Interference); participants' ratings of the number of days in the past week they felt fatigued (0-7; FSI Days); and participants' rating of what percent of each day in the past week, on average, they felt fatigued (0-100; FSI Percent).[75] Individuals scoring at or above the cutoff also report significantly greater scores on these other subscales. Sleep: Sleep will be assessed using the Pittsburgh Sleep Quality Index (PSQI), with a score of >5 considered disturbed sleep as adjusted for cancer populations.[76, 77]
Pain: The Brief Pain Inventory (BPI) assesses pain intensity as well as pain-related interference in function.[78] BPI Pain Severity score ranges from 0-40 (first four items), and the BPI Pain Interference score is a mean of the last 7 items (5a-5g) with a range of 0-10.  
The information gathered from this study will provide important data regarding the impact of CAR-T therapy on patient QOL outcomes.
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guidelines for reporting of depression

Should the patient endorse any thoughts of suicidality or self harm per the IDAS, the study coinvestigator for this portion of the ancillary testing (Dr. Jennifer Knight) will contact them by phone and standard of care referrals will be made. Should completion of the study surveys prompt participants to want treatment for any of the other symptoms, participants will be offered a referral for appropriate care through the Quality of Life Center at the Froedtert Cancer Center.
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12 statistical considerations 12.1 study

There are two primary endpoints in this study—feasibility and safety. Feasibility of CAR-20/19-T cell production by the CliniMACS Prodigy processing device (Miltenyi Biotec) will be measured. At minimum, we expect to reach our target CAR-20/19-T cell dose in at least 75% of patients with an adequate T cell starting dose. Feasibility will be measured at the end of the study after completion of accrual. If >25% of patients with an adequate T cell starting dose were unable to achieve goal CAR-20/19-T cell dose, we would deem that this technique is not a feasible method for production.  
Safety of administration of CAR-20/19-T cells in patients will be assessed as outlined in Section 6 Treatment plan.
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12.2 accrual estimates

This is a Phase 1/1b study evaluating the safety and efficacy CAR-20/19-T cells as part of a first-in-human study. We will utilize a 3+3 escalation design to identify the target dose level followed by a dose expansion at the selected dose level. The minimum number of patients enrolled will be 4 patients (2 at dose level 0 and 2 at dose level -1). The maximum number of patients enrolled would be 24, which is dependent on the number of DLTs experienced on trial.  
Should this treatment be safe and feasible, we would use the identified dose to open a larger phase II study to demonstrate the efficacy of our treatments.
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12.3 interim analyses and stopping

There are no planned interim analyses as part of this Phase 1/1b Study.  
Toxicity will be considered unacceptable and lead to clinical hold of the study if any of the following occur in the first 5 patients: one treatment related mortality (TRM) or three grade 4 non-hematological toxicity events as assessed by PI or DSMB. This ensures that there is only a 5% probability of stopping if the actual TRM is <1%. For Grade 4 non-hematological events not resulting in death, this rule ensures a 5% probability of stopping if the true incidence is <20%
and an 68% probability of stopping if the incidence is 60% or more. Additionally, if there are ≥ 2 DLTs at any dose level, no further patients will be allowed administration at that dose level.  
| 12.4 Statistical Analyses Plans Phase 1 The first 3 patients with NHL or CLL/SLL will be  treated consecutively at Dose Level 0 and will  be monitored for DLTs for a full 28 days. At  each dose level, the first two patients treated   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
In terms of production, if within the first 8 patients with an adequate starting T-cell dose ≥3 patients fail to meet the targeted CAR-20/19-T cell dose, the study will be placed on a clinical hold to re-evaluate production methods and optimize manufacturing. will be treated no sooner than 28 days apart to allow monitoring of acute and sub-acute toxicities. After that patients will be infused no sooner than at 14 day intervals among the specified dose level or in-between two dose levels.  
Dose escalation will proceed within each cohort per the following scheme: A cohort of three patients with NHL or CLL/SLL will receive 2.5 x 105 cells/kg (Dose Level 0). If there are no DLTs in the first three patients, the dose will be escalated to 7.5 x 105 cells/kg (Dose Level 1). If 1/3 patients have a DLT at Dose Level 0, an additional three patients will be enrolled at this dose level. If there is ≤1/6 patients with a DLT, the dose will increase to Dose Level 1. A similar dose escalation schema will be used to escalate from Dose Level 1 to Dose Level 2 (2.5 x 106 cells/kg) which is our goal dose. Dose escalations will NOT occur until all patients at the current level are monitored for 28 days after CAR-20/19-T cell infusion. If two of the first three or six patients with NHL or CLL/SLL develop DLT at Dose Level 0, the dose will de-escalate to 1.0 x 105 cells/kg and accrual will proceed per the same schema as above. If two of the first three or six patients with NHL or CLL have a DLT at the lower dose, the study will be stopped for failure to achieve the desired safety endpoint. The minimum number of patients enrolled will be 4 patients (2 at dose level 0 and 2 at dose level -1) if the first four patients all experience a DLT. The maximum number of patients enrolled in our Dose Escalation phase would be 18.  
Once a safe dose has been identified, the Dose Expansion phase of the study will be open to accrual once approval is obtained from local and federal regulatory bodies. In this Phase, there will be a 6-12 patient expansion cohort in NHL and CLL/SLL. DLT monitoring will continue in the Dose Expansion phase. If ≥2 patients experience a DLT within this expansion cohort, the expansion phase will be placed on hold and will be re-opened only after discussion with the local DSMC and IRB.
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phase 1b

The Phase 1b cohort will be open to accrual once the final subject has enrolled on the Phase 1 portion of the study. This will test the safety of single day CAR-T infusion.
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expansion single infusion

9 patients will be enrolled at the goal dose of 2.5 x 106 cells/kg. The first two patients at this dose level will be observed for 14 days prior to additional enrollment. Among this cohort if there are ≥3 DLTs, this arm will be closed early.
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dose limiting toxicity

The dose limiting toxicity will be based on the tolerability observed during the first 28 days of treatment/observation. If multiple toxicities are seen, the presence of dose limiting toxicity will be based on the most severe toxicity experienced. If a patient dies from a cause felt to be *unrelated* to CAR-T cells (e.g., progressive disease) before completion of the 28 day DLT monitoring period, that patient will be replaced. Patients who experience toxicity from lymphodepletion chemotherapy that prevents administration of CAR-20/19-T cells will also be replaced. All patients who fail to produce the desired dose level will be followed for DLTs, AEs, and survival but will be replaced for this dose-finding and dose-expansion study.  
The definition of DLT for this study is detailed in Section 3.4 The statistical analysis will be primarily descriptive in keeping with the exploratory nature of the study. We will present summary tables of baseline demographic and clinical characteristics of the patients. Adverse events will be tabulated for both overall and within major categories and by grade. Analysis of other secondary endpoints such as anti-tumor activity will be presented as response rates by disease specific sub-type. Statistical support will be provided by Mei-Jie Zhang, PhD the senior statistician on this study. Dr. Zhang will participate in the study design, data analyses, and the scientific manuscript writing associated with this project.
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12.5 analysis population

All patients enrolled and consented for this protocol who underwent CAR-20/19-T cell production will be evaluable for our feasibility endpoint. Only patients who receive CAR-20/19-T cells will be evaluable for our safety endpoint.
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12.6 lost to follow-up

Should a subject fail to return to the clinic for a scheduled protocol specific visit, personnel at the investigational site will need to make 2 attempts by a combination of telephone/e-mail and mail to contact the subject. Sites must document both attempts to contact the subject. If a subject does not respond within 1 month after the second contact the subject will be considered lost to follow-up and no additional contact will be required.
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13 data and safety monitoring

The Medical College of Wisconsin (MCW) Data and Safety Monitoring Committee (DSMC) and the MCW Institutional Review Board (IRB) will approve protocol-specific DSM plans. A local, investigator-initiated trial will be required to be continuously monitored by the principal investigator of the study with monthly (or as each new patient is dosed if no patients enrolled in the month) safety and progress reports submitted to the DSMC. For the purpose of this study, a medical monitor will be appointed to evaluate serious AEs (Grade 3-5) on a real-time basis. This medical monitor is responsible for evaluating serious AEs and can convene a conference call of DSMC chair, DSMC reviewers, or external reviewers when necessary.
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13.1 study team

The study team consists of the investigators, clinical research coordinators/assistants, regulatory specialist, pharmacists, laboratory team, and the study biostatistician.
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13.3 clinical trials office

The MCWCC Clinical Trials Office [CTO] provides administrative assistance and support to the DSMC.
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13.4 dsmc

The Medical College of Wisconsin Cancer Center places the highest priority on ensuring the safety of patients participating in clinical trials. Every cancer interventional trial conducted at MCW includes a plan for safety and data monitoring.  
More information can be found related to the MCWCC Data and Safety Monitoring Plan at the MCWCC website (Data and Safety Monitoring Plan). This study will be reviewed by the Medical College of Wisconsin Cancer Center Data Safety Monitoring Committee (MCW-CC DSMC). External reviewers with CAR-T experience will serve on this committee to provide their expertise. A summary of the MCW CC DSMC activities are as follows:
Review the clinical trial for data integrity and safety  
Review all unexpected grade 3, and all grade 4, and 5 adverse events, as well as any others requiring expedited reporting as defined in this protocol. (Grades 4 & 5 events must be reported to the DSMC within 5 calendar days of study staff's knowledge.) Review all DSM reports Submit a summary of any recommendations related to study conduct Terminate the study if deemed unsafe for patients
A copy of the MCW-CC Data and Safety Monitoring Plan and membership roster will be maintained in the study research file and updated as membership changes. The committee will review reports from the study PI twice annually (or more frequently if needed) and provide recommendations on trial continuation, suspension or termination as necessary. Any available DSMC letters will be submitted to the IRB of record as required.
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14 regulatory compliance ethics and

This study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki as stated in 21 CFR §312.120(c)(4); consistent with GCP and all applicable regulatory requirements.
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14.2 regulatory compliance

This study will be conducted in compliance with:
The protocol  
Federal regulations, as applicable, including: 21 CFR 50 (Protection of Human Subjects/Informed Consent); 21 CFR 56 (Institutional Review Boards) and §312 (Investigational New Drug Application; and 45 CFR 46 Subparts A (Common Rule), B (Pregnant Women, Human Fetuses and Neonates), C (Prisoners), and D (Children),
GCP/ICH guidelines, and all applicable regulatory requirements. The IRB must comply with the regulations in 21 CFR §56 and applicable regulatory requirements.
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14.3 institutional review board

The protocol, the proposed informed consent form and all forms of participant information related to the study (e.g., advertisements used to recruit participants) will be reviewed and approved by the MCW Institutional Review Board. Prior to obtaining MCW approval, the protocol must be approved by the Medical College of Wisconsin Cancer Center Scientific Review Committee. The initial protocol and all protocol amendments must be approved by the IRB prior to implementation. The MCW IRB is AAHARPP accredited.
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informed consent process

Informed consent is a process that is initiated prior to the individual's agreeing to participate in the study and continues throughout the individual's study participation. Extensive discussion of risks and possible benefits of this therapy will be provided to the subjects and their families. Consent forms describing in detail the study interventions/products, study procedures and risks are given to the subject and written documentation of informed consent is required prior to starting intervention/administering study product.  
Informed consent from the patient will be obtained using forms approved by the local institution's IRB. Informed consent will be obtained by the Principal Investigator or CoInvestigators. Consent forms will be IRB-approved and the subject will be asked to read and review the document. Upon reviewing the document, the investigator will explain the research study to the subject and answer any questions that may arise. In accordance with 46 CR 46.111, the subject or representative legal guardian will sign and date the informed consent document prior to any procedures being done specifically for the study. The subjects may withdraw consent at any time throughout the course of the trial.  
A copy of the informed consent document will be given to the subjects for their records. The MCWCC CTO will follow the MCW/FH IRB's policy for subjects who demonstrate limited English proficiency or limited literacy. The original consent is kept with the subject's study file, and a copy of the consent is sent to the OCRICC office, which will then submit to HIM a copy of the signed consent to be scanned into EPIC, the legal medical record.
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14.4 subject confidentiality and access

Subject confidentiality is strictly held in trust by the sponsor-investigator, participating investigators, and any staff. This confidentiality includes the clinical information relating to participating subjects, as well as any genetic or biological testing. The study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor-investigator.  
The conditions for maintaining confidentiality of the subjects' records are required for the life of the data. These rules apply equally to any and all MCWCC projects.  
While data are being collected and after all data have been collected but are still in the process of being analyzed, the subject's data/PHI are stored in the locked Clinical Research office in the Clinical Trials Office. Databases in which the study subject information is stored and accessed are password protected, allowing for limited access by authorized personnel only. Data/PHI kept in the Case Report Forms in Oncore contain the study identifiers, subject initials, date of birth and date of service.  
The principal investigator will allow access to all source data and documents for the purposes of monitoring, audits, IRB review, and regulatory inspections. The Clinical Trials Office quality assurance monitor may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the subjects in this study. The clinical study site will permit access to such records.
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onsite audits

Auditing is essential to ensure that research conducted at the Medical College of Wisconsin (MCW) Cancer Center is of the highest quality and meets MCW and regulatory agency standards. Regulatory authorities, the IRB and/or sponsor may request access to all source documents, data capture records and other study documentation for on-site audit or inspection. Direct access to these documents must be guaranteed by the investigator, who must provide support at all times for these activities.
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14.5 quality assurance

This protocol will be reviewed internally every six months by the Cancer Center Clinical Trials Office Quality Assurance Staff. Approximately 30% of subject files will be selected randomly for review (max 10 subjects at each monitoring timepoint). Consent, eligibility and objective based data will be reviewed for those files selected. One file will be selected randomly for a comprehensive review at each quality assurance review timepoint. Regulatory will be reviewed at the time of each review.
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14.6 changes in the protocol

Once the protocol has been approved by the MCW IRB, any changes to the protocol must be documented in the form of an amendment. The amendment must be signed by the investigator and approved by IRB prior to implementation. If it becomes necessary to alter the protocol to eliminate an immediate hazard to patients, an amendment may be implemented prior to IRB approval. In this circumstance, however, the Investigator must then notify the IRB in writing within five working days after implementation. The IRB may provide, if applicable regulatory authority(ies) permit, expedited review and approval/favorable opinion for minor change(s) in ongoing studies that have the approval
/favorable opinion of the IRB. The investigator will submit all protocol modifications to the sponsor and the regulatory authority(ies) in accordance with the governing regulations.  
Any departures from the protocol must be fully documented in the source documents.
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15 data handling and record

Every effort is made to uphold the integrity of the project, the research, the institution, and the researchers involved. Data collection guidelines and methodologies are carefully developed before the research begins. Investigators focus on the following to ensure data integrity: welltrained data collectors/recorders to ensure consistency and quality, well-designed data collection protocols and ongoing monitoring. In this way, study rigor and validity are maintained.  
Data is protected from physical damage as well as from tampering, loss or theft. This project's data management is a multidisciplinary activity that includes investigators, research coordinators and nurses, data mangers, support personnel, biostatisticians and database programmers. Quality control will be applied to each stage of data handling to ensure that all data are reliable and have been processed correctly. MCW Cancer Center database will be utilized for data collection and case report forms through OnCore.
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15.2 study record retention

The principal investigator will maintain adequate records of the disposition of the CAR-20/19-T cells, including dates, quantity and use by subjects and the disposition of cells manufactured but not infused.  
The principal investigator, along with assistance from the research coordinators, is required to prepare and maintain adequate and accurate case histories that record all observations and other data pertinent to the investigation on each individual administered the CAR-20/19-T cells. Case histories include the case report forms and supporting data including, for example, signed and dated consent forms and medical records including, for example, progress notes of the physician, the individual's hospital chart(s), and the nurses' notes. The case history for each individual shall document that informed consent was obtained prior to participation in the study.  
Study documentation includes all CRFs, data correction forms or queries, source documents, sponsor-investigator correspondence, monitoring logs/letters, and regulatory documents (e.g., protocol and amendments, IRB correspondence and approval, signed patient consent forms).  
Source documents include all recordings of observations or notations of clinical activities and all reports and records necessary for the evaluation and reconstruction of the clinical research study.  
In accordance with FDA regulations, the investigator shall retain records for a period of two years following the date a marketing application is approved for the drug for the indication for which it is being investigated; or, if no application is to be filed or if the application is not approved for such indication, until two years after the investigation is discontinued and FDA is notified.
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15.3 publishing data

Data from this study will be managed by the clinical trials office at MCW and the investigators listed on this study. Upon completion of the study, the data will be submitted to national meeting followed by a plan to publish in peer-reviewed journal.
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16 references

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669-73.  
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figure 1 study schema
appendix a performance status criteria

| Karnofsky Performance Scale   |                                                                                                               |
|-------------------------------|---------------------------------------------------------------------------------------------------------------|
| Percent                       | Description                                                                                                   |
| 100                           | Normal, no complaints, no evidence  of disease                                                                |
| 90                            | Able to carry on normal activity; minor  signs or symptoms of disease Normal activity with effort; some signs |
| 80                            | or symptoms of disease Cares for self, unable to carry on                                                     |
| 70                            | normal activity or to do active work Requires occasional assistance, but                                      |
| 60                            | is able to care for most of his/her  needs                                                                    |
| 50                            | Requires considerable assistance and  frequent medical care                                                   |
| 40                            | Disabled, requires special care and  assistance  Severely disabled, hospitalization                           |
| 30                            | indicated Death not imminent                                                                                  |
| 20                            | Very sick, hospitalization indicated Death not imminent                                                       |
| 10                            | Moribund, fatal processes  progressing rapidly                                                                |
| 0                             | Dead                                                                                                          |
figure 1 study schema
appendix b neurological assessment

The Mini-Mental State Examination will be performed as per Table 1: Schedule of Events.
figure 1 study schema
appendix c 2004 mas/hlh diagnostic

Diagnosis of MAS/HLH requires meeting 5/8 following criteria
- Fever
- Splenomegaly - Cytopenias (affecting at least 2 of 3 lineages in the peripheral blood)
o Hemoglobin <9 g/dL
o Platelets <100,000/µL o Absolute neutrophil count<1000/µL
- Hypertriglyceridemia and/or hypofibrinogenemia:
o Fasting triglycerides >265 mg/dl o Fibrinogen <1.5 g/L
- Hemophagocytosis in bone marrow or spleen or lymph nodes
- Low or absent NK-cell activity (according to local laboratory reference)
- Ferritin >500 ng/mL
- Soluble CD25 (i.e., soluble IL-2 receptor) >2,400 U/ml
appendix d schema for testing
day -6 in-process testing (minimal

(*Rationale*: For those receiving CAR-T cells as a fresh infusion, lymphodepleting chemotherapy would be initiated on Day -4 This allows 2 days to verify CAR-T potency before lymphodepletion is initiated.)
1. Sterility cultures 2. Transduction efficiency (Protein L staining for CD20_19 expression)
3. Basic phenotyping (CD3, CD4, CD8 from Protein L panel)
4. Viability (from Protein L panel) and viable cell numbers 5. Chromium release assays versus CD19+/ CD20+ Raji target cells (potency)
6. Mycoplasma testing (in-house) 7. Endotoxin testing (sendout)
appendix d schema for testing
day 0 testing of final

(*Important Note*: For those receiving CAR-T cells as a fresh infusion, results of gram stains, flow cytometry (to assess transduction efficiency) and 7-AAD viability will be required for product release.)
1. Sterility cultures 2. Gram stain 3. Transduction efficiency (by Protein L staining) 4. Comprehensive phenotyping 5. Chromium release assays versus CD19+/ CD20+ Raji target cell and transfected CD19+
and CD20+ target cells (potency)
6. Intracellular cytokine staining assays (potency) 7. VCN & VSVg testing (sendout) 8. Sendout for final mycoplasma and endotoxin testing
appendix d schema for testing
appendix e car-t cell treatment

□ **Tumor Lysis Prevention**
o Allopurinol 300 mg daily for tumor lysis prevention starting Day -4 until Day+141
□ **Seizure prophylaxis**
o Keppra (Levetiracetam) 500 mg twice daily from Day -4 until Day +28
□ **Prophylactic Antibiotics**
o PCP prophylaxis to be started Day -4 through Day+28 (agent and dosing per institutional standards)
o Anti-viral prophylaxis: Acyclovir or valacyclovir starting Day -4 through Day+28. Valganciclovir or IV ganciclovir can be given as a substitute as indicated by treating provider.  
o Neutropenic prophylaxis: As per institutional standards, not mandatory
(e.g. ciprofloxacin or cefepime)
o Fungal prophylaxis as per treating physician, not mandatory
□ **Pre-medications for CAR-20/19-T cells (1 hour prior to CAR-T cell infusion)**
o Acetaminophen 650 mg x 1 orally o Diphenhydramine hydrochloride 50 mg x 1 oral or IV2 Transfusion Parameters o Maintain Platelets>20 K/µL and Hemoglobin>8.0 mg/dL
o Filgrastrim can be given at the discretion of the treating provider starting Day +21, provided the patient does not have signs/symptoms consistent with CAR-T cell neurotoxicity or cytokine release syndrome.  
1- Can be stopped at discretion of treating physician 2- Alternatives anti-histamines in place of diphenhydramine hydrochloride can be given per discretion of treating physician (e.g. cetirizine, ranitidine) Dosed per institutional standards.
appendix f assessment/management of cytokine
appendix e car-t cell treatment

| Grade                                                               | Toxicity                                                            | High-dose vasopressors (all doses are required for ≥3   |                              |
|---------------------------------------------------------------------|---------------------------------------------------------------------|---------------------------------------------------------|------------------------------|
| hours)                                                              |                                                                     |                                                         |                              |
| Grade 1                                                             | Symptoms are not life threatening and require symptomatic treatment |                                                         |                              |
| only, eg, fever, nausea, fatigue, headache, myalgias, malaise       | Pressor                                                             | Dose                                                    |                              |
| Grade 2                                                             | Symptoms require and respond to moderate intervention               | Norepine monotherapy                                    | ≥ 20 µg/min                  |
| Oxygen requirement <40% or                                          | Dopamine monotherapy                                                | ≥ 10 µg/kg/min                                          |                              |
| Hypotension responsive to fluids or low dose² of one vasopressor or | Phenylephrine monotherapy                                           | ≥ 200 µg/min                                            |                              |
| Grade 2 organ toxicity                                              | Epinephrine monotherapy                                             | ≥ 10 µg/min                                             |                              |
| Grade 3                                                             | Symptoms require and respond to aggressive intervention             | If on vasopressin                                       | Vasopressin + norepinephrine |
| Oxygen requirement ≥ 40% or                                         | equivalent of ≥ 10 µg/min*                                          |                                                         |                              |
| Hypotension requiring high dose* or multiple vasopressors or        | Norepinephrine equivalent of                                        |                                                         |                              |
| If on combination vasopressors                                      |                                                                     |                                                         |                              |
| Grade 3 organ toxicity or grade 4 transaminitis                     | (not vasopressin)                                                   | ≥ 20 µg/min*                                            |                              |
| Grade 4                                                             | Life-threatening symptoms                                           |                                                         |                              |
| Requirement for ventilator support or                               |                                                                     |                                                         |                              |
| Grade 4 organ toxicity (excluding transaminitis)                    |                                                                     |                                                         |                              |
| Grade 5                                                             | Death                                                               |                                                         |                              |  
1. Lee, D.W., et al., Current concepts in the diagnosis and management of cytokine release syndrome. Blood, 2014. 124(2): p. 18895.  
| Table 5: CRS Symptoms, Evaluation, Management, Response CRS assessed  via Lee et al.  Revised CRS  grading system.  Grade 1  toxicities Grade 2  toxicities1 Grade 3 or Grade 4 toxicities Hypotension that  responds to fluid or one  low dose pressor, oxygen  requirement<40%, Grade  2 organ toxicity per  CTCAE 5.0 Fevers, myalgias,  Symptoms nausea/fatigue, malaise,  headache Hypotension requiring high dose or multiple  pressors, Grade 3 or 4 organ toxicity, life  threatening symptoms, mechanical ventilation Infectious work-up,  initiation/escalation of  antibiotics per institutional  standards. IV fluids, antipyretic therapy as  indicated with cooling  blankets and  acetaminophen (avoid  NSAIDs). Supportive care  measure2 Admission to hospital (if  not admitted). IV fluids,  infectious work-up,  supportive care measures Admission to hospital, intensive care unit transfer as indicated. IV fluids, infectious  work-up, respiratory support, and renal  replacement therapy as indicated Evaluation &  Management Persistent symptoms  >12-24 hours despite  interventions or  escalation to Grade 3-4  toxicity initiate CRS  directed treatment3 Persistent Grade 1  symptoms>24-48 hours  or escalation to Grade 3-4  toxicities initiate CRS  directed treatment3 Response &  Escalation Immediate initiation of CRS directed  treatments3   |
|---|  
1-At direction of treating physician, CRS treatment can be initiated in any patient with Grade≥2 toxicity 2-Recommended Supportive care measure listed in Appendix G 3-CRS Directed Treatment Algorithm is on Page 91
appendix f assessment/management of cytokine
table 6 neurotoxicity evaluation and

| Neurotoxicity assessed via  CTCAE 5.0                                                                                                                     | Grade 1                                                                                                                                                                                                                                | Grade 2                                                                                                   | Grade 3   | Grade 4   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------|-----------|-----------|
| Moderate memory  impairment or moderate  lethargy limiting ADL.  Moderate weakness or  sensory loss moderate  neuropathy limiting  instrumental ADL, etc. | Severe memory  impairment limiting selfcare, severe weakness  or sensory loss, multiple  seizures, severe  neurological deficit, etc.                                                                                                                                                                                                                                        |                                                                                                           |           |           |
| Mild memory  impairment, reduced  awareness or alertness,  mild paresthesia, etc.                                                                         | Life threatening  consequence, cerebral  edema1 urgent  intervention indicated                                                                                                                                                         |                                                                                                           |           |           |
| Examples                                                                                                                                                  | Neurological Exam,                                                                                                                                                                                                                     |                                                                                                           |           |           |
| Evaluation                                                                                                                                                | additional work-up as  indicated                                                                                                                                                                                                       | Neurological Examination, MRI Brain +/- lumbar puncture and EEG as  indicated. Neurological consultation. |           |           |
| Admission to hospital (if  not admitted), neurochecks Q4 hours,  supportive care, work-up  as above.  Dexamethasone can be  given at discretion of  treating physician                                                                                                                                                           | Admission to Intensive Care Unit. Dexamethasone  10 mg IV/PO every 6-12 hours (max dose 10 mg  Q6 hours). If not improving consider tocilizumab 8  mg/kg IV x 1 (max dose 800 mg). Can give up to 2  doses of Tocilizumab in 24 hours. |                                                                                                           |           |           |
| Clinical vigilance,                                                                                                                                       |                                                                                                                                                                                                                                        |                                                                                                           |           |           |
| Management                                                                                                                                                | monitoring, supportive  care                                                                                                                                                                                                           |                                                                                                           |           |           |
| 1-For patients with evidence of cerebral edema (Grade 4 toxicity) the following specific interventions will be performed - Intensive Care Unit care       |                                                                                                                                                                                                                                        |                                                                                                           |           |           |  
1-For patients with evidence of cerebral edema (Grade 4 toxicity) the following specific interventions will be performed
- Intensive Care Unit care
- Neurosurgical consultation
- Mechanical ventilation - Pharmacological support per intensivist and neurological and neurosurgical recommendations
cytokine release syndrome directed treatment[56]
table 6 neurotoxicity evaluation and

With Neurological Toxicities1  
  
| Continue Dexamethasone until  significant improvement + supportive  care as per Appendix G. Patients with  progressive systemic CRS symptoms   |
|------------------------------------------------------------------------------------------------------------------------------------------------|  
1-Patients with neurological toxicities (Grade≥3 by CTCAE 5.0) will be treated with dexamethasone with or without the presence of systemic CRS.
cytokine release syndrome directed treatment[56]
appendix g supportive care measures

Guidelines developed and adapted from *Brudno, J. N. and J. N. Kochenderfer (2016).*
"Toxicities of chimeric antigen receptor T cells: recognition and management." Blood 127*(26):*
3321-3330.
cytokine release syndrome directed treatment[56]
table 7 supportive care guidelines

| Table 7: Supportive Care Guidelines for Patients Receiving CAR-T cells Constitutional - Administer acetaminophen for symptomatic management of fevers in patients  with normal hepatic function.  - Provide cooling blankets for fevers >40o C  - Avoid corticosteroids and NSAIDs - Avoid meperidine  Cardiovascular - Minimize anti-hypertensives prior to cell infusion  - Monitor vital signs at least every 4 hours for up to 48 hours' post CAR-20/19-T  cell infusion - Monitor vital signs Q2H in patients with fevers and tachycardia  - Initiate replacement of IV fluids for patients with poor oral intake or high  insensible losses to maintain net even fluid balance - IV fluid boluses for patients with SBP less than their pre-infusion baseline or  those with hypotension SBP<90  - Persistent hypotension not responsive to fluids should be initiated on  vasopressors and patients transferred to intensive care unit  - For patient on vasopressors evaluate cardiac function with troponin and ECG.  Echocardiogram can be performed as indicated.  Infectious disease - Prophylactic antibiotics as per Appendix E - Neutropenic Fever management as per institutional guidelines with broad  spectrum antibiotics. Appropriate fever work-up with chest x-ray, urinalysis,  and blood cultures  Hematologic - Initiate Allopurinol for TLS prevention as per Appendix E - Transfuse RBCs for Hgb<8 g/dL - Transfuse platelets for Platelets<20 k/µL - Monitor ANC as per schedule of events (Table 1). Neupogen can be started at  Day+21 in patients with persistent neutropenia as per guidelines in Section 6.9 - Transfuse fresh frozen plasma in patients with PTT prolongation >2.0 fold  upper limit of normal  - Transfuse cryoprecipitate to maintain fibrinogen ≥100 mg/dL Neurologic - Neuro checks Q4H by nursing staff in patients with ≥Grade 2 neurotoxicity - MRI Brain +/- lumbar puncture and EEG in patients with ≥Grade 2  neurotoxicity  - Neurological consultation for patients with ≥Grade 2 neurotoxicity  - MMSE evaluation as per Table 1   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
cytokine release syndrome directed treatment[56]
appendix h operating procedures for

Some patients will have successful production of CAR-20/19-T cells but fail to make the adequate cell dose as required per our protocol. Those patients will be allowed treatment but required to be replaced for the purposes of this Phase 1/1b study. Patients will be followed as per this clinical protocol with monitoring for DLT, safety, adverse events, and long-term survival. Specific Criteria for Infusion of Non-Specified CAR-20/19-T cell dose 1) Meet all eligibility criteria a listed in Section 4 2) Meet all release criteria for CAR-T cell product 3) Meet same infusion criteria as listed in Section 6.3 and 7.1 4) Patients will be followed per schedule of events listed in Section 5 and Table 1
cytokine release syndrome directed treatment[56]
analysis of patients treated with

At first, a maximum of 3 patients will be allowed to be treated in this manner. If there is ≤1 DLT within the first 28 days for the first three patients, further patients will be allowed study drug with CAR-20/19-T cells even if they don't make the required treatment dose. After enrollment of the 4th patient or beyond in this cohort, if the DLT rate exceeds ≥33%, no further patients will be allowed treatment outside of a specified cell dose per the clinical protocol. All patients will be followed for AEs, survival, response rates as per Table 1.
appendix i quality of life
analysis of patients treated with

QOL MEASURES  
  
  
  
Participant \# _____
IDAS
Instructions: Below is a list of feelings, sensations, problems, and experiences that people sometimes have. Read each item to determine how well it describes your recent feelings and experiences. Then select the option that best describes **how** much you have felt or experienced things this way during the past two weeks, including today.  
| 1. I was proud of myself                                  | 1   | 2   | 3   | 4   | 5   |
|-----------------------------------------------------------|-----|-----|-----|-----|-----|
| 2. I felt exhausted                                       | 1   | 2   | 3   | 4   | 5   |
| 3. I felt depressed                                       | 1   | 2   | 3   | 4   | 5   |
| 4. I felt inadequate                                      | 1   | 2   | 3   | 4   | 5   |
| 5. I slept less than usual                                | 1   | 2   | 3   | 4   | 5   |
| 6. I felt fidgety, restless                               | 1   | 2   | 3   | 4   | 5   |
| 7. I had thoughts of suicide   8. I slept more than usual | 1   | 2   | 3   | 4   | 5   |
| 9. I hurt myself purposely                                | 1   | 2   | 3   | 4   | 5   |
| 10. I slept very poorly                                   | 1   | 2   | 3   | 4   | 5   |
| 11. I blamed myself for things                            | 1   | 2   | 3   | 4   | 5   |
| 12. I had trouble falling asleep                          | 1   | 2   | 3   | 4   | 5   |
| 13. I felt discouraged about things                       | 1   | 2   | 3   | 4   | 5   |
| 14. I thought about my own death                          | 1   | 2   | 3   | 4   | 5   |  
| 15. I thought about hurting myself                                                                   | 1   | 2   | 3   | 4   | 5   |
|------------------------------------------------------------------------------------------------------|-----|-----|-----|-----|-----|
| 16. I did not have much of an appetite                                                               | 1   | 2   | 3   | 4   | 5   |
| 17. I felt like eating less than usual                                                               | 1   | 2   | 3   | 4   | 5   |
| 19. I did not feel much like eating                                                                  | 1   | 2   | 3   | 4   | 5   |
| 21. I felt optimistic                                                                                | 1   | 2   | 3   | 4   | 5   |
| 23. I felt that I had accomplished a lot                                                             | 1   | 2   | 3   | 4   | 5   |
| 24. I looked forward to things with enjoyment                                                        | 1   | 2   | 3   | 4   | 5   |
| 25. I was furious                                                                                    | 1   | 2   | 3   | 4   | 5   |
| 26. I felt hopeful about the future                                                                  | 1   | 2   | 3   | 4   | 5   |
| 27. I felt that I had a lot to look forward to                                                       | 1   | 2   | 3   | 4   | 5   |
| 28. I felt like breaking things   29. I had disturbing thoughts of something bad that happened to me | 1   | 2   | 3   | 4   | 5   |
| 30. Little things made me mad                                                                        | 1   | 2   | 3   | 4   | 5   |
| 31. I felt enraged                                                                                   | 1   | 2   | 3   | 4   | 5   |
| 32. I had nightmares that reminded me of something bad that happened                                 | 1   | 2   | 3   | 4   | 5   |
| 33. I lost my temper and yelled at people                                                            | 1   | 2   | 3   | 4   | 5   |
| 34. I felt like I had a lot of interesting things to do.                                             | 1   | 2   | 3   | 4   | 5   |
| 35. I felt like I had a lot of energy                                                                | 1   | 2   | 3   | 4   | 5   |
| 36. I had memories of something scary that happened                                                  | 1   | 2   | 3   | 4   | 5   |
| 37. I felt self-conscious knowing that others were watching me                                       | 1   | 2   | 3   | 4   | 5   |  
| 38. I felt a pain in my chest                               | 1   | 2   | 3   | 4   | 5   |
|-------------------------------------------------------------|-----|-----|-----|-----|-----|
| 39. I was worried about embarrassing myself socially        | 1   | 2   | 3   | 4   | 5   |
| 40. I felt dizzy or light headed                            | 1   | 2   | 3   | 4   | 5   |
| 41. I cut or burned myself on purpose                       | 1   | 2   | 3   | 4   | 5   |
| 42. I had little interest in my usual hobbies or activities | 1   | 2   | 3   | 4   | 5   |
| 43. I thought that the world would be better off without me | 1   | 2   | 3   | 4   | 5   |
| 44. I felt much worse in the morning than later in the day  | 1   | 2   | 3   | 4   | 5   |
| 45. I felt drowsy, sleepy                                   | 1   | 2   | 3   | 4   | 5   |
| 46. I woke up early and could not get back to sleep         | 1   | 2   | 3   | 4   | 5   |
| 47. I had trouble concentrating                             | 1   | 2   | 3   | 4   | 5   |
| 48. I had trouble making up my mind                         | 1   | 2   | 3   | 4   | 5   |
| 49. I talked more slowly than usual                         | 1   | 2   | 3   | 4   | 5   |
| 50. I had trouble waking up in the morning                  | 1   | 2   | 3   | 4   | 5   |
| 51. I found myself worrying all the time                    | 1   | 2   | 3   | 4   | 5   |
| 52. I woke up frequently during the night                   | 1   | 2   | 3   | 4   | 5   |
| 53. It took a lot of effort for me to get going             | 1   | 2   | 3   | 4   | 5   |
| 54. I woke up much earlier than usual                       | 1   | 2   | 3   | 4   | 5   |
| 55. I was trembling or shaking                              | 1   | 2   | 3   | 4   | 5   |
| 56. I became anxious in a crowded public setting            | 1   | 2   | 3   | 4   | 5   |
| 57. I felt faint                                            | 1   | 2   | 3   | 4   | 5   |  
| 58. I found it difficult to make eye contact with people         | 1   | 2   | 3   | 4   | 5   |
|------------------------------------------------------------------|-----|-----|-----|-----|-----|
| 59. My heart was racing or pounding                              | 1   | 2   | 3   | 4   | 5   |
| 60. I got upset thinking about something bad that happened       | 1   | 2   | 3   | 4   | 5   |
| 61. I found it difficult to talk with people I did not know well | 1   | 2   | 3   | 4   | 5   |
| 62. I had a very dry mouth                                       | 1   | 2   | 3   | 4   | 5   |
| 63. I was short of breath                                        | 1   | 2   | 3   | 4   | 5   |
| 64. I felt like I was choking                                    | 1   | 2   | 3   | 4   | 5   |
appendix i quality of life
fsi

Instructions: The following questions ask about fatigue during the **past week**. For each of the following, circle the one number that best indicates how that item applies to you.  
| 0                                                                                                      | 1                | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9   | 10   |    |
|--------------------------------------------------------------------------------------------------------|------------------|-----|-----|-----|-----|-----|-----|-----|-----|------|----|
| Not at all                                                                                             | As fatigued as   |     |     |     |     |     |     |     |     |      |    |
| fatigued                                                                                               | I could be       |     |     |     |     |     |     |     |     |      |    |
| 2. Rate your level of fatigue on the day you felt least fatigued during the past week. 0 1 2 3 4 5 6 7 | 8                | 9   | 10  |     |     |     |     |     |     |      |    |
| Not at all                                                                                             | As fatigued as I |     |     |     |     |     |     |     |     |      |    |
| fatigued                                                                                               | could be         |     |     |     |     |     |     |     |     |      |    |
| 3. Rate your level of fatigue on the average in the last week. 0 1 2 3 4 5                             | 6                | 7   | 8   | 9   | 10  |     |     |     |     |      |    |
| Not at all                                                                                             | As fatigued as I |     |     |     |     |     |     |     |     |      |    |
| fatigued                                                                                               | could be         |     |     |     |     |     |     |     |     |      |    |
| 4. Rate your level of fatigue right now.  0 1 2 3                                                      | 4                | 5   | 6   | 7   | 8   | 9   | 10  |     |     |      |    |
| Not at all                                                                                             | As fatigued as I |     |     |     |     |     |     |     |     |      |    |
| fatigued                                                                                               | could be         |     |     |     |     |     |     |     |     |      |    |
| 5. Rate how much, in the past week, fatigue interfered with: No                                        | Extreme          |     |     |     |     |     |     |     |     |      |    |
| interference                                                                                           | interference     |     |     |     |     |     |     |     |     |      |    |
| a. Your general level of activity                                                                      | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| b. Your ability to bathe and dress yourself                                                            | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| c. Your normal work activity (includes both  work outside the home and housework)                      | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| d. Your ability to concentrate                                                                         | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| e. Your relations with other people                                                                    | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| f. Your enjoyment of life                                                                              | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| g. Your mood                                                                                           | 0                | 1   | 2   | 3   | 4   | 5   | 6   | 7   | 8   | 9    | 10 |
| 6. Indicate how many days, in the past week, you felt fatigued for any part of the day:                |                  |     |     |     |     |     |     |     |     |      |    |  
0 1 2 3 4 5 6 7 7. Rate **how much of the day**, on average, you felt fatigued in the past week 0 1 2 3 4 5 6 7 8 9 10 None of the day The entire day
appendix i quality of life
psqi

Instructions: The following questions relate to your usual sleep habits during the **past month only**. Your answers should indicate the most accurate reply for the majority of days and nights in the past month. Please answer all questions.  
1. During the **past month**, what time have you usually gone to bed at night?  
BED TIME ___________
2. During the **past month,** how long (in minutes) has it usually taken you to fall asleep each night?  
NUMBER OF MINUTES ___________
3. During the **past month**, what time have you usually gotten up in the morning?  
GETTING UP TIME ___________
4. During the **past month**, how many hours of actual sleep did you get at night? (This may be different than the number of hours you spent in bed.)
HOURS OF SLEEP PER NIGHT ___________
For each of the remaining questions, check the one best response. Please answer all questions.  
5. During the **past month**, how often have you had trouble sleeping because you . . .  
a) Cannot get to sleep within 30 minutes  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
b) Wake up in the middle of the night or early morning  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
c) Have to get up to use the bathroom  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
d) Cannot breathe comfortably  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
e) Cough or snore loudly  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
f) Feel too cold j) Other reason(s), please describe_________________________________________________
__________________________________________________________________________
How often during the past month have you had trouble sleeping because of this?  
| Not during the                    | Less than         | Once or twice   | Three or more      |
|-----------------------------------|-------------------|-----------------|--------------------|
| past month _____                  | once a week _____ | a week _____    | times a week _____ |
| g) Feel too hot  Not during the   | Less than         | Once or twice   | Three or more      |
| past month _____                  | once a week _____ | a week _____    | times a week _____ |
| h) Had bad dreams  Not during the | Less than         | Once or twice   | Three or more      |
| past month _____                  | once a week _____ | a week _____    | times a week _____ |
| i) Have pain Not during the       | Less than         | Once or twice   | Three or more      |
| past month _____                  | once a week _____ | a week _____    | times a week _____ |  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
6. During the **past month**, how would you rate your sleep quality overall?  
| Very good   | __________   |
|-------------|--------------|
| Fairly good | __________   |
| Fairly bad  | __________   |
| Very bad    | __________   |  
7. During the **past month**, how often have you taken medicine to help you sleep (prescribed or
"over the counter")?  
Not during the past month _____  
Less than once a week _____  
Once or twice a week _____  
Three or more times a week _____  
8. During the **past month**, how often have you had trouble staying awake while driving, eating meals, or engaging in social activity?  
Not during the  
  
past month _____
Less than once a week _____
Once or twice a week _____
Three or more times a week _____
9. During the **past month**, how much of a problem has it been for you to keep up enough enthusiasm to get things done?  
No problem at all __________ Only a very slight problem __________
Somewhat of a problem __________
A very big problem __________
appendix i quality of life
bpi

Instructions: The following questions ask about pain during the **past week**. For each of the following, circle the one number that best indicates how that item applies to you.  
1. Please rate your pain by circling the number that best describes your pain at its **worst** in the last week.  
0 1 2 3 4 5 6 7 8 9 10 No pain Pain as bad as you can imagine 2. Please rate your pain by circling the number that best describes your pain at its **least** in the last week.  
0 1 2 3 4 5 6 7 8 9 10 No pain Pain as bad as you can imagine 3. Please rate your pain by circling the number that best describes your pain on the **average** in the last week.  
0 1 2 3 4 5 6 7 8 9 10 No pain Pain as bad as you can imagine 4. Please rate your pain by circling the number that best describes your pain **right now**.  
0 1 2 3 4 5 6 7 8 9 10 No pain Pain as bad as you can imagine  
| 5. Circle the number that describes how, during the past week, pain has interfered with your:  Does not interfere a. General activity 0 1 2 3 4 5 6   | 7   | 8   | 9   | 10   |    |    |    |    |    |    |    |
|-------------------------------------------------------------------------------------------------------------------------------------------------------|-----|-----|-----|------|----|----|----|----|----|----|----|
| b. Mood                                                                                                                                               | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |
| c. Walking ability                                                                                                                                    | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |
| d. Normal work (includes both work outside the  home and housework)                                                                                   | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |
| e. Relations with other people                                                                                                                        | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |
| f. Sleep                                                                                                                                              | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |
| g. Enjoyment of life                                                                                                                                  | 0   | 1   | 2   | 3    | 4  | 5  | 6  | 7  | 8  | 9  | 10 |  
THESE QUESTIONS NEED ONLY BE ANSWERED ONCE; NO NEED TO ANSWER AT SUBSEQUENT STUDY FOLLOW-UP VISITS: Please indicate below what best represents your household income:
_____ <$10,000
_____ $10,001-$25,000 _____ $25,001-$40,000 _____ $40,001-$55,000 _____ $55,001-$70,000 _____ $70,001-$85,000 _____ $85,001-$100,000 _____ > $100,000 Please indicate which education level best describes you: _____ Less than 12 years
_____ High School
_____ Trade School _____ Some College
_____ College Graduate _____ Post-Graduate Degree
appendix i quality of life
appendix j specimen collection and

For patients who have progressed, mandatory blood samples for monitoring for CAR-T cell persistence which include T-cell persistence by flow and by quantitative PCR and presence of replication competent virus by Q-PCR for VSV-g can be drawn locally and sent to Medical College of Wisconsin for analysis.
appendix i quality of life
peripheral blood sample

- T-cell Persistent Studies: A minimum of 20mL of peripheral blood, separated into 2-10mL EDTA
(purple top) tubes.  
- Q-PCR Samples: A minimum of 20mL of peripheral blood, separated into 2- 10mL EDTA (purple top) tubes.  
- VSV-g Samples: A minimum of 20mL of peripheral blood, separated into 2- 10mL EDTA (purple top) tubes.
appendix i quality of life
specimen labeling

All submitted specimens must be labeled with the patient's initials, patient study ID\#, patient's date of birth and date/time of specimen collection. (All supplies and airbills will be provided in research sample kit).
appendix i quality of life
shipping requirements

All specimens must be accompanied by a Specimen Shipping Requisition Form. Samples must be received by the Medical College of Wisconsin within 24 hours of draw. The Froedtert-Cell Processing Lab is open M-F, 8:00am - 4:30 pm. Please do not draw samples on Fridays for delivery on Saturday. The sample must be placed in a leak proof primary receptacle (ex: vacutainer tube). Multiple primary receptacles must be individually wrapped or separated to prevent contact. The primary receptacle must be placed into a leak proof secondary container (ex: resealable biohazard bag) in such a way that under normal conditions of transport, they cannot break or leak.  
Absorbent material, such as paper towels or absorbent pads, must be placed in the secondary container with sufficient capacity to absorb the entire contents of the primary receptacle(s).  
The secondary container must be placed into an outer shipping container with suitable cushioning material. (ie: Gel pack) Ice packs to be added to the bottom of shipping container, and on top of the samples. The shipping container must be labeled with the universal biohazard symbol.  
The outer packaging must be marked with the name, address, and phone number of both the sender and recipient. All packages shipped via aircraft must display a 2-inch diamond with "UN3373" inside of the diamond.  
Send the container via next day delivery at refrigerated temperature to:
Froedtert- Cell Processing Lab Attn: Fenlu Zhu 9200 W. Wisconsin Ave Milwaukee, WI 53226 Please call/email the Cell Processing Lab and Coordinator as soon as the tracking number is available:
(414)805-6144, Fenlu Zhu:FZhu@mcw.edu, and (414)805-8378, Sharon Yim: syim@mcw.edu, The Cell Processing Lab is open M-F, 8:00am - 4:30 pm. Please do not draw samples on Fridays for delivery on Saturday.