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clinical study protocol

Phase 1/1b Study of Redirected Autologous T Cells Engineered to Contain an Anti CD19 and Anti CD20 scFv Coupled to CD3ζ and 4-1BB Signaling Domains in Patients with Relapsed and/or Refractory CD19 or CD20 Positive B Cell Malignancies
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short protocol title

Phase 1/1b Study of CAR-20/19-T Cells in Patients with Relapsed Refractory B Cell Malignancies
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proprietary and confidential

The information in this document is considered privileged and confidential, and may not be disclosed to others except to the extent necessary to obtain Institutional Review Board approval and informed consent, or as required by federal and state laws. Persons to whom this information is disclosed should be informed that this information is privileged and confidential and that it should not be further disclosed.
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mcw protocol no. pro00028724. fda

Nirav Shah, MD MS
Assistant Professor of Medicine Medical College of Wisconsin 9200 W. Wisconsin Ave Milwaukee, WI 53226 Phone: 414-805-4600 Fax: 414-806-6815 Email address: nishah@mcw.edu Co-Principal Investigator Parameswaran Hari, MD MS Professor Hematology/Oncology Medical College of Wisconsin 9200 W. Wisconsin Ave Milwaukee, WI 53226 Phone: 414-805-4600 Fax: 414-806-6815 Email: phari@mcw.edu Biostatistician:
Mei-Jie Zhang, PhD Professor Division of Biostatistics Medical College of Wisconsin 9200 W. Wisconsin Ave Milwaukee, WI 53226 Email: meijie@mcw.edu
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co-investigator

Timothy S. Fenske MD
Professor of Medicine Medical College of Wisconsin 9200 W. Wisconsin Ave Milwaukee, WI 53226 Phone: 414-805-4600 Fax: 414-806-6815 tfenske@mcw.edu Principal Lab Investigator Bryon D. Johnson, PhD
Professor of Medicine, Hem/Onc Director MCW BMT Laboratories Medical College of Wisconsin 8700 Watertown Plank Road Milwaukee, WI, 53226 Phone 414-805-6145 Email: bjohnson@mcw.edu
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co-lab investigator

Fenlu Zhu, PhD Assistant Professor of Medicine Medical College of Wisconsin 8700 Watertown Plank Road Milwaukee, WI, 53226 Phone 414-805-6145 fzhu@mcw.edu
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co-investigator

Mehdi Hamadani MD Professor of Medicine Medical College of Wisconsin 9200 W. Wisconsin Ave Milwaukee, WI 53226 Phone: 414-805-4600 Fax: 414-806-6815 mhamdani@mcw.edu
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protocol summary

| Title                                                                                                                                                                             | Phase 1/1b Study of Redirected Autologous T Cells Engineered to  Contain an Anti CD19 and Anti CD20 scFv Coupled to CD3ζ and 4- 1BB Signaling Domains in Patients with Relapsed and/or Refractory  CD19 or CD20 Positive B Cell Malignancies                                                                                                                                                                                                                            |                                                                                                                                                                                             |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Protocol Number                                                                                                                                                                   | PRO00028724                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                             |
| IND #                                                                                                                                                                             | 17518                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                             |
| Principal Investigator                                                                                                                                                            | Nirav Shah, MD MSHP Assistant Professor, Department of Medicine                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                             |
| Study Sites                                                                                                                                                                       | Froedtert Hospital & Medical College of Wisconsin Cancer Center                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                             |
| Clinical Trial Phase                                                                                                                                                              | Phase 1/1b                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                             |
| Study Disease                                                                                                                                                                     | CD19 or CD20 Positive B Cell Malignancies including CLL/SLL and  B-cell NHL                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                             |
| Inclusion Criteria                                                                                                                                                                | 1.                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Diagnosis of B-cell NHL or CLL/SLL: Patients must be aged≥18  years with relapsed, refractory disease and no available curative  options that meet clinical criteria to initiate treatment. |
| 2.                                                                                                                                                                                | Patients with B-cell NHL or CLL/SLL must have either CD19 or  CD20 positive disease on most recent biopsy performed (a  repeat biopsy is not mandatory for this study except as noted  below). A minimum of 5% CD19 or CD20 positivity by  immunohistochemistry or flow cytometry on prior or repeat  biopsy is required.                                                                                                                                               |                                                                                                                                                                                             |
| 3.                                                                                                                                                                                | Absolute CD3+ T cell count ≥50/mm3                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                             |
| 4.                                                                                                                                                                                | MRI brain and Lumbar Puncture with CSF analysis by cytology  and flow cytometry without evidence of CNS involvement ONLY  in patients with history of CNS involvement                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                             |
| 5.                                                                                                                                                                                | Measurable disease must have been documented within 4 weeks  of the time of consent defined as the following by disease specific  subtype: a. B-cell NHL: Active disease defined as nodal lesions greater  than 20 mm in the long axis or extranodal lesions >10 mm in  long and short axis or bone marrow involvement that is biopsy  proven b. CLL/SLL: Active disease by either bone marrow, peripheral  flow cytometry, or CT and/or PET imaging with nodal disease |                                                                                                                                                                                             |
| 6.                                                                                                                                                                                | Patients should have failed at least two                                                                                                                                                                                                                                                                                                                                                                                                                                | lines of a standard                                                                                                                                                                         |
| treatment and meet disease specific criteria detailed below:                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                             |
| a.                                                                                                                                                                                | CLL/SLL: measurable disease as defined above that has relapsed  after at least one line of chemo-immunotherapy and progressed  or intolerant to ibrutinib monotherapy                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                             |
| b.                                                                                                                                                                                | CD19 or CD20                                                                                                                                                                                                                                                                                                                                                                                                                                                            | positive B cell NHL limited to the following                                                                                                                                                |
| histologies: Advanced Stage III or IV Follicular Lymphoma,  Diffuse Large B cell Lymphoma and associated subtypes (e.g.  aggressive B-cell lymphoma, T-cell/histocyte rich B-cell |                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                             |  
| lymphoma, primary mediastinal B-cell lymphoma, EBV+ diffuse  large B-cell lymphoma, transformed lymphoma such as  transformed follicular or marginal zone lymphoma, and Richter's  transformation) and Mantle cell lymphoma. Specific criteria  include: - Patients must have active, measurable disease after two lines  of cytotoxic chemotherapy of which one must be anthracycline  containing.  - Must have received Rituximab or another CD20 antibody and  at minimum two chemotherapy regimens appropriate for their  disease.  - Either failed autologous transplant or ineligible to receive  autologous transplant                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                 |                                                                                 |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------|
| 7.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Karnofsky performance score ≥70. See Appendix A for scales.                                                                                                                                                                                                                                                     |                                                                                 |
| 8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Normal Baseline Neurological Evaluation: Mini-Mental Status  Exam Score 24-30 (Appendix B)                                                                                                                                                                                                                      |                                                                                 |
| 9.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Adequate hepatic function, defined as AST and ALT <5 x upper  limit of normal (ULN); serum bilirubin and alkaline phosphatase  <5 x ULN, or considered not clinically significant as per the clinical  PIs discretion (e.g. Gilbert's or indirect hyperbilirubinemia) or felt  to be due to underlying disease. |                                                                                 |
| 10. Adequate renal function, defined as creatinine clearance>60  ml/min 11. Able to provide written informed consent  12. Agree to practice birth control during the study 13. Adequate cardiac function as indicated by New York Heart  Association (NYHA) classification I or II AND left ventricular  ejection fraction of ≥35% (by cardiac ECHO or MUGA) and  adequate pulmonary function as indicated by room air oxygen  saturation of ≥92%. 14. Expected survival >12 weeks 15. Negative urine or serum pregnancy test in females of child bearing  potential at study entry and again within 48 hours' prior  lymphodepleting chemotherapy. 16. Patients with prior blinatumomab treatment require repeat biopsy  post-blinatumomab treatment that demonstrates CD19 or CD20  positive disease.  17. Meet criteria for regarding fertility and contraception detailed in  section 4.4 18. Central line access will be required for CAR-20/19-T cell infusion. |                                                                                                                                                                                                                                                                                                                 |                                                                                 |
| Exclusion Criteria                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | 1.                                                                                                                                                                                                                                                                                                              | Positive beta-HCG in female of child-bearing potential defined as  per table 1. |
| 2.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Patients with known systemic allergy to bovine or murine  products.                                                                                                                                                                                                                                             |                                                                                 |
| 3.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Known prior positive serology for human anti-mouse antibody  (HAMA).                                                                                                                                                                                                                                            |                                                                                 |
| 4.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Confirmed active human immunodeficiency virus (HIV), Hepatitis  B or C infection.                                                                                                                                                                                                                               |                                                                                 |
| 5.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | History of significant autoimmune disease OR active, uncontrolled  autoimmune phenomenon: such as systemic lupus erythematous,                                                                                                                                                                                  |                                                                                 |
| IIT-CAR-T-Shah                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | 4                                                                                                                                                                                                                                                                                                               | Version No.: 5                                                                  |  
| Wegner's glomerulonephritis, autoimmune hemolytic anemia,  idiopathic thrombocytopenic purpura (AIHA, ITP) requiring steroid  therapy defined as >20 mg of prednisone or equivalent daily.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                       |                |            |               |      |      |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|------------|---------------|------|------|
| 6.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Presence of ≥grade 3 non-hematologic toxicities as per CTCAE  version 5.0 from any previous treatment unless it is felt to be due  to underlying disease.                                                                                                                                                                                                                             |                |            |               |      |      |
| 7.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Concurrent use of investigational therapeutic agents or enrollment  on another therapeutic clinical trial at any institution. Minimum of  ≥4 weeks required from administration of any other investigational  agents on other clinical trials prior to enrollment on this CAR-T  protocol.                                                                                            |                |            |               |      |      |
| 8.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Refusal to participate in the long-term follow-up protocol                                                                                                                                                                                                                                                                                                                            |                |            |               |      |      |
| 9.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Patients with active CNS involvement by malignancy on MRI or  by lumbar puncture. a. Patients with prior CNS disease that has been effectively  treated will be eligible providing treatment was >4 weeks  before enrollment and a remission documented within 8  weeks of planned CAR-T cell infusion by MRI brain and CSF  analysis.                                                |                |            |               |      |      |
| 10. Previous                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | recipients                                                                                                                                                                                                                                                                                                                                                                            | of             | allogeneic | hematopoietic | stem | cell |
| transplantation (AHCT) are excluded if they are <100 days' posttransplant, have evidence of active graft-versus-host-disease  (GVHD) of any grade, or are currently on immunosuppression.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                                                                       |                |            |               |      |      |
| 11. Previous CAR-T cell therapy directed at either CD19 or CD20 within 100 days of planned CAR-20/19-T cell infusion (does not  include re-enrollment) a. Patients with prior CAR-T treatment against CD19 or CD20  must have repeat biopsy confirming a minimum of 5% CD19  or CD20 positivity by immunohistochemistry or flow  cytometry 12. Anti-CD20 antibody treatment within 4 weeks of cell infusion 13. Anti-CD19 antibody treatment within 4 weeks of cell infusion 14. Cytotoxic chemotherapy other than lymphodepletion within 14  days of CAR-T cell infusion  15. Cytotoxic chemotherapy treatment within 14 days or steroid  treatment (other than replacement dose steroids) within 7 days  prior to apheresis collection for CAR-T cells 16. Patients post solid organ transplant who develop high grade  lymphomas or leukemias 17. Concurrent active malignancy other than basal or squamous cell  carcinomas of the skin |                                                                                                                                                                                                                                                                                                                                                                                       |                |            |               |      |      |
| Study Rationale                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | The study is divided into two phases. Phase 1 and Phase 1b.  Phase 1: To evaluate a novel closed processing method for the  production of the first dual CD20 and CD19 CAR-T cell and  determine the safety of administration of these cells as part of a first  in human study consisting of 2 cohorts; dose escalation and dose  expansion. The CAR-T cells were given over 2 days. |                |            |               |      |      |
| IIT-CAR-T-Shah                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | 5                                                                                                                                                                                                                                                                                                                                                                                     | Version No.: 5 |            |               |      |      |  
| Phase 1b: To determine the safety of single dose (unfractionated  CAR-T cell infusion at 2.5 x 106 cells/kg   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                     |
|---------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| Primary Objectives                                                                                            | 1.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Determine the safety (dose level) of CAR-20/19-T cell  administration in relapsed refractory CLL/SLL and B-cell NHL |
| 2.                                                                                                            | Determine the feasibility to manufacture CAR-20/19-T cells  locally from patient apheresis products using the CliniMACS  Prodigy Cell processing device                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                     |
| 1.                                                                                                            | Determine the anti-tumor responses as measured by response  rates.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                     |
| 2.                                                                                                            | Describe the duration of response for responding patients                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                     |
| 3.                                                                                                            | Determine persistence of CAR-20/19-T cells                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                     |
| 4.                                                                                                            | Determine the effects of CAR-20/19-T infusion on non-neoplastic  CD19 & CD20 B cells in vivo                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                     |
| 5.                                                                                                            | Determine if cellular or humoral host immunity develops against  CAR-20/19-T cells                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                     |
| 6.                                                                                                            | Evaluate MRD using molecular technologies                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                     |
| Secondary  Objectives Endpoints                                                                               | 1. Ability to manufacture CAR-20/19-T cells from patient apheresis  products using the CliniMACS Prodigy processing device (Miltenyi  Biotec). The number T cell lines that do not meet criteria for T cell  purity, total transduced T cell content, viability, and sterility will be  determined.  2. Occurrence of adverse events, defined as either CRS related  Grade 3/4 toxicity or other NCI CTCAE version 5.0 non-hematologic  ≥ grade 3 signs/symptoms, laboratory toxicities and clinical events  that are possibly, probably or definitely related to study treatment at  any time from the infusion until day +28 post CAR-T infusion                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                     |
| Study Design                                                                                                  | This is a phase 1/1b, interventional, single arm, open label, treatment  study designed to evaluate the safety and feasibility of infusion of  CAR-20/19-T in patients with B cell malignancies that have relapsed  after prior therapies and are not candidates for curative intent  standard therapy.  The study is divided into two phases. Phase 1 and Phase 1b.  Phase 1: consists of 2 cohorts; dose escalation and dose  expansion. The CAR-T cells were given over 2 days.  In the phase 1 portion, there will be a dose escalation cohort to  determine the safe CAR-20/19-T cell dose in patients with CLL/SLL  and NHL. Once the desired dose has been identified there will be a  6 patient dose expansion phase at the specified dose level.  Phase 1b: CAR-T infusion given as a single dose.  In the Phase 1b portion of the study, we will test the safety of  unfractionated CAR-T cells utilizing the safe dose identified in the  phase 1 portion (2.5 x 106  cells/kg).  CAR-20/19-T cells will be administered either fresh or thawed after  cryopreservation by IV injection. Patients will receive one of four  dose levels of CAR-20/19-T cells based on our dose escalation  design (Section 6). Cells will be given over 2 days, 30% on Day 0 |                                                                                                                     |
| Study Agent  Description                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                     |  
| and 70% on Day 1 in the Phase 1 portion and in Phase 1b as a  single infusion on Day 0. Cell dose will be maxed at 80 kg.  Phase 1 Dose Level -1: 1 x 105 CAR-20/19-T cells/kg  Dose Level 0: 2.5 x105 CAR-20/19-T cells/kg (starting dose level) Dose Level 1: 7.5 x105 CAR-20/19-T cells/kg  Dose Level 2: 2.5 x106 CAR-20/19-T cells/kg (goal cell dose) Phase 1b Expansion Dose Level: 2.5 x 106  cells/kg (single infusion)   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Number of Subjects                                                                                                                                                                                                                                                                                                                                                                                                                 | A maximum of 24 patients will be enrolled on this Phase 1/1b study.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |
| Subject Participation                                                                                                                                                                                                                                                                                                                                                                                                              | Subjects will be followed for up to 2 years post infusion of the CAR20/19-T cells                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| Duration Duration of Follow up                                                                                                                                                                                                                                                                                                                                                                                                     | Patients will be followed for up to 2 years post infusion of CAR-20/19- T cells infusion as part of this protocol and then placed on a longterm follow-up protocol that will follow patients for up to a total of 15  years post CAR-T cell infusion                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| Estimated Time to                                                                                                                                                                                                                                                                                                                                                                                                                  | 18-24 months                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Complete Enrollment: Statistical                                                                                                                                                                                                                                                                                                                                                                                                   | This is a phase 1/1b, interventional single arm, open label,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Methodology:                                                                                                                                                                                                                                                                                                                                                                                                                       | treatment study designed to evaluate the safety and feasibility of  infusion of CAR-20/19-T cells. In the phase 1 portion the study will  utilize a 3+3 dose escalation design in NHL & CLL/SLL followed by  a 6 patient expansion cohort. In the Phase 1b portion, 9 patients will be treated at the safe dose  from the Phase 1 portion (2.5 x 106  cells/kg).and will receive a  single infusion rather than fractionated dose.                                                                                                                                                                                                                                                            |
| Safety Assessments                                                                                                                                                                                                                                                                                                                                                                                                                 | Safety will be determined by assessments of cytokine release  syndrome (CRS) using the Lee et al. grading scale [1] and adverse  events via NCI CTCAE version 5.0. Dose limiting toxicities (DLT) will  be monitored for the first 28 days after CAR-20/19-T cell infusion as  per Table 1. DLT will be defined a grade 3-4 non-hematologic toxicity  possibly, probably, or definitely related to the infusion of CAR-20/19- T cells. All deaths (grade 5 toxicity) in the first 28 days felt to be either  possibly, probably, or definitely related to CAR-20/19-T cell therapy  will be considered a DLT. CRS related DLTs and hematologic DLT  are defined in the protocol (section 3.4) |
| Efficacy                                                                                                                                                                                                                                                                                                                                                                                                                           | CLL/SLL & B-cell NHL at Day +28 (+/- 3 days)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Assessments                                                                                                                                                                                                                                                                                                                                                                                                                        | PET/CT or CT neck/chest/abd/pelvis + BM Aspirate and Biopsy                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |  
| This is the first phase 1/1b study to evaluate the safety and efficacy  of a CAR-T cell therapy directed against two B cell antigens and  produced under GMP conditions using the closed system CliniMACS Prodigy device in B cell malignancies.   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Unique Aspects of  this Study                                                                                                                                                                                                                      |
figure 1 study schema
table 1 schedule of events

| ᶲ2 to 4  days prior to  day -4                                    | #Day +2  to Day +7   | $Follow  up  (Year  1-2)   |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
|-------------------------------------------------------------------|----------------------|----------------------------|--------|-------|-----------------------------|------|-------|-------|-------|-------|-------|-------|-------|-------|--------|----|----|----|----|
| Week -8                                                           | Day                  |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| to                                                                |                      |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Day -15                                                           | -15                  | ♦Day  -4  LDP              | §Day 0 | Day   |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| +6  weeks                                                         | +1                   | Day                        | Day    | Day   | Day                         | Day  | Day   | Day   | Day   | Day   | Day   | Day   |       |       |        |    |    |    |    |
| +10                                                               | +14                  | +21                        | +28    | +60   | +90                         | +120 | +150  | +180  | +270  | +365  |       |       |       |       |        |    |    |    |    |
| Visit Window                                                      | Screening/           | Aphere                     | +/- 1  | +/- 5 | CAR-20/19-T  cells infusion |      | +/- 1 | +/- 2 | +/- 2 | +/- 3 | +/- 7 | +/- 7 | +/- 7 | +/- 7 | +/- 14 |    |    |    |    |
| Enrollment                                                        | sis                  | day                        | days   | day   | days                        | days | days  | days  | days  | days  | days  | days  | +/-   | +/- 1 | +/- 1  |    |    |    |    |
| 14                                                                | month                | month                      |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| days                                                              |                      |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Signed Inform Consent                                             | x                    |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Apheresis                                                         | x                    |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| CAR-20/19-T cells infusion1                                       |                      | x                          | x1     |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Confirmation of Diagnosis of CD19  or CD20 positive B-cell NHL or | x                    |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| CLL/SLL2 Recent History and Physical  Examination 3               | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     | x      | x  | x  | x  | x  |
| Karnofsky performance status                                      | x                    | x                          | x      | x     |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Vitals4                                                           | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     | x      | x  | x  | x  | x  |
| Concomitant Medication &  Evaluation of Adverse Events            | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     | x      | x  | x  | x  | x  |
| Bone Marrow Transplant Markers 5                                  | x                    | x                          | x      | x     | x                           | x    | x     |       |       |       |       |       |       |       |        |    |    |    |    |
| Tumor Measurements by PET/CT,  CT Scans6                          | x                    | x6                         | x      | x     | x                           | x    |       |       |       |       |       |       |       |       |        |    |    |    |    |
| ECG7                                                              | x                    | x                          | x      |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| ECHO/MUGA                                                         | x                    |                            |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Lumber Puncture 8                                                 | x                    | x8                         |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| MRI Brain9                                                        | x                    | x9                         |        |       |                             |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| Bone Marrow Biopsy & Aspirate 10-11                               | x                    | x                          | x      | x     | x                           |      |       |       |       |       |       |       |       |       |        |    |    |    |    |
| CBC and Differential                                              | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     | x      | x  | x  | x  |    |
| Comprehensive Metabolic Panel12                                   | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     | x      | x  | x  | x  |    |
| LDH/Uric Acid                                                     | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     | x     | x     | x     |        |    |    |    |    |
| Quantitative Immunoglobulins                                      | x                    | x                          | x      | x     | x                           | x    | x     | x     | x     | x     | x     |       |       |       |        |    |    |    |    |  
| ᶲ2 to 4  days prior to  day -4                                | #Day +2  to Day +7   | $Follow  up  (Year  1-2)   |         |       |                             |      |       |       |       |       |       |       |       |       |       |
|---------------------------------------------------------------|----------------------|----------------------------|---------|-------|-----------------------------|------|-------|-------|-------|-------|-------|-------|-------|-------|-------|
| Week -8  to                                                   | Day -15              |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Day -15                                                       | ♦Day  -4  LDP        | §Day 0 +6                  | Day  +1 |       |                             |      |       |       |       |       |       |       |       |       |       |
| weeks                                                         | Day                  | Day                        | Day     | Day   | Day                         | Day  | Day   | Day   | Day   | Day   | Day   |       |       |       |       |
| +10                                                           | +14                  | +21                        | +28     | +60   | +90                         | +120 | +150  | +180  | +270  | +365  |       |       |       |       |       |
| +/-                                                           |                      |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Visit Window                                                  | Screening/           | Aphere                     | +/- 1   | +/- 5 | CAR-20/19-T  cells infusion |      | +/- 1 | +/- 2 | +/- 2 | +/- 3 | +/- 7 | +/- 7 | +/- 7 | +/- 7 | 14    |
| Enrollment                                                    | sis                  | day                        | days    | day   | days                        | days | days  | days  | days  | days  | days  | days  | +/-   | +/- 1 | +/- 1 |
| 14  days                                                      | month                | month                      |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Ferritin/CRP/ESR                                              | x                    | x                          | x       | x     | x                           | x    | x     | x     |       |       |       |       |       |       |       |
| PT/INR/PTT/D-Dimer/Fibrinogen                                 | x                    | x                          | x       | x     | x                           | x    | x     | x     |       |       |       |       |       |       |       |
| TSH/Free T4                                                   | x                    | x                          | x       |       |                             |      |       |       |       |       |       |       |       |       |       |
| Lipid Panel                                                   | x                    | x                          |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Urinalysis                                                    | x                    | x                          | x       |       |                             |      |       |       |       |       |       |       |       |       |       |
| Beta-2-Microglobulin                                          | x                    | x                          |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Autoimmune Screen (ANA)                                       | x                    |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Pregnancy Test (serum or urine) 13                            | x                    | x                          |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Infectious Disease Markers 14                                 | x                    |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Respiratory Virus Panel 15                                    |                      | x                          |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Creatinine Clearance 16                                       | x                    |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Central Line17                                                | x                    |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| Research Studies                                              |                      |                            |         |       |                             |      |       |       |       |       |       |       |       |       |       |
| T Cell Persistent Studies 18                                  |                      | x                          | x       | x     | x                           | x    | x     | x     | x     | x     | x     | x     |       |       |       |
| Q-PCR for integrated vector DNA19                             |                      | x                          | x       | x     | x                           | x    | x     |       |       |       |       |       |       |       |       |
| Presence of replication competent  virus by Q-PCR for VSV-g20 | x                    | x                          | x       | x     | x                           | x    |       |       |       |       |       |       |       |       |       |
| Cytokine Studies) 21                                          |                      | x                          | x       | x     | x                           | x    | x     | x     |       |       |       |       |       |       |       |
| Neurological evaluations22                                    | x                    | x                          | x       | x     |                             |      |       |       |       |       |       |       |       |       |       |
| T-cell repertoire Diversity Studies23                         | x                    | x                          | x       | x     |                             |      |       |       |       |       |       |       |       |       |       |
| PaxGene RNA Tubes25                                           | x                    | x                          | x       | x     |                             |      |       |       |       |       |       |       |       |       |       |
| Tryptophan Metabolites25                                      | x                    | x                          | x       | x     |                             |      |       |       |       |       |       |       |       |       |       |
| Quality of Life Studies26                                     | x                    | x                          | x       | x     | x                           | x    | x     |       |       |       |       |       |       |       |       |
figure 1 study schema
footnotes

♦ Start of Lymphodepleting Chemotherapy (LDP) Theses assessments should be completed daily during each day of LDP unless otherwise specified. Start date of LDP chemotherapy will be determined on the date of CAR-T cell infusion.  
ᶲ Pre-lymphodepletion visit will be based on date of CAR-T cell administration and hence can move forward by up to 6 weeks
§ CAR-20/19-T cells may be given fresh after manufacturing without cryopreservation or can be cryopreserved and given up to 6 weeks after production \# Daily from Days +1 through Day +7 unless otherwise specified in Section 5.4.6 $ Clinical Visits every 6 months (+/- 1 month) from Year 1 to Year 2 1- Split-dosing of CAR-T cells through Phase 1 escalation/expansion. Single dosing of CAR-T cells in Phase 1b 2- Patients will need documentation of relapsed or refractory B cell malignancies. CD19 or CD20 positivity ≥5% by IHC or Flow cytometry required on one biopsy.  
Please see eligibility criteria for specific requirements for each patient.  
3- History and height only required at screening. Physical exam, weight at all other time points 4- Vitals should include Blood Pressure, Temperature, Respiratory rate, Pulse & Pulse Ox. On the days of CAR-20/19-T cell infusion, vitals should be done prior to infusion, at the end of infusion, and every 15 minutes thereafter for a minimum of one-hour post infusion.  
5- Absolute numbers of CD3+ T cells, CD4+ T cells and CD8+ T cells should be assessed before scheduling for apheresis collection to determine protocol eligibility. Minimum of ≥50 CD3+ T cells/mm3 required, with a mixture of both CD4+ and CD8+ T cells.  
6- Diagnostic imaging is required at baseline (within 8 weeks of CAR-20/19-T cell infusion) and at 1 month post-cell infusion (PET/CT preferred). Diagnostic CT
scans of the neck/chest/abdomen/pelvis must be performed if PET/CT scans cannot be obtained at baseline due to timing constraints, insurance or reimbursement issues, or other reasons. Either PET/CT or diagnostic CT scans are required to document response. If >8 weeks' elapse from date of imaging and planned T-cell infusion, repeat imaging will be required. For follow-up patients with get either PET/CT or diagnostic CT scans at Day +28, 6 months, and 1 year. For year 1-2 scans will be done yearly.  
7- ECG to be performed prior to CAR-20/19-T cell infusion on Day 0 8- Lumbar Puncture with CSF analysis by cytology and flow cytometry is required at screening/eligibility for all patients with prior history of CNS disease. If >8 weeks elapse from last LP and planned CAR-20/19-T cell infusion, a repeat LP will be required in patients with a history of CNS disease and who exhibit CNS
symptoms.  
9- MRI Brain will be performed only for patients with history of prior CNS disease or signs/symptoms of active CNS disease. MRI will be repeated if >8 weeks elapse between previous MRI and scheduled CAR-20/19-T cell infusion.  
10- Patients should have a staging bone marrow biopsy and aspirate ≤ 8 weeks prior to CAR-20/19-T cell infusion. If >8 weeks' elapse, a repeat biopsy will be indicated.  
11- All patients will have a repeat marrow at Day +28 post-CAR-T cell infusion. Beyond Day +28, for patients with B-cell NHL ongoing marrow biopsy core and aspirate at 6 months, 1, year, and 2 year is indicated only if positive at baseline. Patients with CLL/SLL are required to have BM biopsy at all specified timepoints (6 months, 1 year, 2 year). Bone marrow biopsy and aspirate should also be obtained at time of suspected relapse. If baseline marrow is positive only by flow cytometry, the aspirate and flow cytometry should be repeated.  
12- Comprehensive Metabolic Panel: Albumin, Alkaline Phosphatase, AST, Bicarb, Bili T, Calcium (total), Chloride, Creatinine, Glucose, Potassium (K), Sodium
(NA), Protein Total, BUN, ALT.  
13- Within 48 hours prior to the start of lymphodepleting chemotherapy. Definition of female of childbearing potential(FCBP): A FCBP is a sexually mature female regardless of sexual orientation or whether she has undergone a tubal ligation who: 1) has not undergone a hysterectomy or bilateral oophorectomy or 2) has not been naturally postmenopausal (amenorrhea following cancer therapy does not rule out childbearing potential) for at least 24 consecutive months (i.e., has had menses at any time in the preceding 24 consecutive months).  
14- Patients must be tested for HIV, Hepatitis B surface antigen/antibody and core antibody, and HCV within 8 weeks prior to CAR T cell infusion. If the HCV
antibody is positive, a screening HCV RNA by any RT-PCR or bDNA assay must be performed. Repeat testing is NOT required.  
15- Viral Respiratory NAAT will be done 2-4 days prior to initiation of lymphodepleting (LDP) chemotherapy if patients exhibit symptoms concerning for viral infection (cough, cold, fever). In the setting of a positive result, initiation of LPD chemotherapy and CAR-T treatment will be delayed until resolution of symptoms.  
16- Calculate Creatinine (by Cockcroft-Gault) at screening. 17- Central Line is required prior to initiation of LDP chemotherapy. 18- T-cell persistent studies: baseline sample to be drawn prior to CAR-20/19-T cell infusion on Day 0 followed by Day +1, +7, +10, +14, +21, +28 and then per calendar above. If there are two consecutive negative tests—**this will not need to be repeated.** Additional samples (blood, CSF, ascites, tissue, etc.) for Tcell persistence can be performed per the investigators discretion as felt to be clinically indicated.  
19- Q-PCR for Integrated Vector DNA: Will be done prior to infusion then at Day +28, 3 months, 6 months, 1 year, 1.5 years, 2 years. If there are two consecutive negative tests—this will not need to be repeated.  
20- Replication competent lentivirus (RCL) will be monitor by Q-PCR for VSV-G (Vesicular stomatitis virus-G) which is a surrogate for RCL. Will be done prior to infusion then at Day +28, 3 months, 6 months, 1 year, 1.5 years, 2 years. Testing will continue until Q-PCR for integrated vector DNA is negative for two consecutive timepoints.  
21- At minimum, will include assays for the following cytokines: IFN-ϒ, IL10, IL6 and TNFα. Additional cytokines will be monitored at discretion of investigators Cytokines will be checked on Day 0, +1, Day +7, Day+10, Day+14, Day+21, Day+28. Additional samples may be taken if indicated based on clinical presentation.  
22- Given concern for neurotoxicity with CAR-T cells, neurological evaluation will be done at baseline and on Day+1, Day+7 and every third day beyond if the patient develops neurological toxicity. For patients without neurological toxicity after CAR-T therapy, re-evaluation will occur on Day+28. Neurological Assessment Tool is found in Appendix B.  
23- T-cell receptor diversity studies. Sample 1 at Week -4 will be from apheresis product. Sample 2 will be from the CAR-T cell product. Sample 3 and 4 will be a peripheral blood sample (10 ml) on Day +7 and Day +28 24- Pax Gene tubes will be collected and stored until sent for analysis as described in Section 11.2 until disease progression 25- Red top tube will be collected at the noted time points and delivered to Dr. Hillard's MCW lab for analysis until disease progression 26- Quality of Life studies until disease progression VISITS REQUIRED AT MCW: All visits through Day+28 will be required to occur at MCW. For responding patients, beyond Day +28, patients will have required visits at MCW 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.  
For patients who progress, follow-up will be as per Section 5.4.7  
| TABLE OF CONTENTS PROTOCOL SUMMARY 3 FIGURE 1: STUDY SCHEMA 9 TABLE 1: SCHEDULE OF EVENTS 10 TABLE OF CONTENTS 14 LIST OF ABBREVIATIONS 18 1 INTRODUCTION 22 1.1 BACKGROUND 22 1.2 INVESTIGATIONAL AGENT(S) 23 1.2.1 FIGURE 3—GENE MAP AND ANIMATION OF THE CD20/CD19 VECTOR 24 1.3 PRECLINICAL DATA 25 1.4 MCW PRECLINICAL DATA 25 1.5 CLINICAL DATA 31 1.6 RATIONALE FOR LYMPHODEPLETION AND CAR-T CELL DOSING 32 1.7 KNOWN AND POTENTIAL RISKS AND BENEFITS 33 2 HYPOTHESIS AND OBJECTIVES 36 2.1 HYPOTHESIS 36 2.2 PRIMARY OBJECTIVES 36 2.3 SECONDARY OBJECTIVES 36 2.4 RATIONALE FOR THE OUTCOME MEASURES SELECTION 36 3 STUDY DESIGN 37 3.1 GENERAL DESCRIPTION 37 3.2 NUMBER OF SUBJECTS 37 3.3 PRIMARY ENDPOINT(S) 37 3.4 DOSE LIMITING TOXICITY 38 3.5 SECONDARY ENDPOINT(S) 39 3.6 STUDY TIMELINE 39 3.7 PRIMARY COMPLETION 39 3.8 STUDY COMPLETION 40 4 PATIENT SELECTION 40 4.1 ELIGIBILITY CRITERIA 40 4.2 INCLUSION CRITERIA 40 4.3 EXCLUSION CRITERIA 41 4.4 SPECIAL CRITERIA FOR REGARDING FERTILITY AND CONTRACEPTION 42 4.5 SUBJECT RECRUITMENT AND SCREENING 42 5 STUDY ENTRY AND WITHDRAWAL; STUDY PROCEDURES 43 5.1 STUDY ENTRY PROCEDURES 43 5.2 PRETREATMENT PERIOD 43 5.3 ENROLLMENT 44 5.4 STUDY PROCEDURES RELATED TO TREATMENT 45 5.4.1 STUDY PROCEDURES - DAY -15 (APHERESIS DAY) 45 5.4.2 MONONUCLEAR CELL APHERESIS 45 5.4.3 STUDY PROCEDURES WEEK -1 + 6 WEEKS (~2 TO 4 DAYS PRIOR TO LYMPHODEPLETION) ...45IIT-CAR-T-Shah 14 Version No.: 5   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| 5.4 S TART OF L YMPHODEPLETION C HEMOTHERAPY (D Y -4 + 6 WEEKS) .......................................46                                                            |    |                |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|----------------|
| 5.4.5 CAR-20/19-T Cell Infusion (+ 6 weeks) ............................................................................46                                           |    |                |
| 5.4.6 Day +1 through day +7 after cell infusion ....................................................................47                                               |    |                |
| 5.4.7 Follow-Up Visits (Day+10 to 2 years) ................................................................................47                                        |    |                |
| 5.5 Study Withdrawal Procedures ..............................................................................................49                                     |    |                |
| 6 TREATMENT PLAN .                                                                                                                                                   |    |                |
| 6.1 Lymphodepletion (within 1 week prior to CAR-20/19-T cell infusion) ........................50                                                                    |    |                |
| 6.2 Preparation of CAR-20/19-T Cell Infusion .............................................................................50                                         |    |                |
| 6.3 Administration of CAR-20/19-T cell infusion on Day 0 and Day +1 (Phase 1) or Day 0                                                                               |    |                |
| (Phase 1b)                                                                                                                                                           |    |                |
| 6.4 Dose Escalation Schedule .                                                                                                                                       |    |                |
| 6.5 Dose Expansion Phase .                                                                                                                                           |    |                |
| 6.6 Phase 1b (Single Dose of CAR-T cell) ..................................................................................................57                        |    |                |
| 6.7 T REATMENT / D OSE L IMITING T OXICITY .......................................................................................................57                 |    |                |
| 6.8 U SAGE OF C ONCURRENT / C ONCOMITANT M EDICATIONS .........................................................................58                                    |    |                |
| 6.9 D IETARY R ESTRICTIONS                                                                                                                                           |    |                |
| 6.10 Prohibited Medications                                                                                                                                          |    |                |
| 6.11 M ONITORING S UBJECT C OMPLIANCE                                                                                                                                |    |                |
| 6.12 Re-treatment                                                                                                                                                    |    |                |
| 7 DOSING DELAYS/DOSE MODIFICATIONS .                                                                                                                                 |    |                |
| 7.1 Dosing Administration/Delays                                                                                                                                     |    |                |
| 7.2 Monitoring and Toxicity Management ............................................................................................60                                |    |                |
| 7.3 O THER T OXICITIES                                                                                                                                               |    |                |
| 8ADVERSE EVENTS: DEFINITIONS AND REPORTING REQUIREMENTS .............................62                                                                              |    |                |
| 8.1 D EFINITIONS .                                                                                                                                                   |    |                |
| 8.2 Unanticipated Problem Involving Risk to Subject or Other (UPIRSO) .....................63                                                                        |    |                |
| 8.3 AE Attribution and Grading ........................................................................................................63                            |    |                |
| 8.4 Monitoring and Recording an Adverse Event  .......................................................................64                                             |    |                |
| 8.5 Procedure for Reporting Drug Exposure during Pregnancy and Birth Events....64                                                                                    |    |                |
| 8.6 S UBJECT C OMPLAINTS .............................................................................................................................65             |    |                |
| 8.7 Routine Reporting Procedures for AEs ...............................................................................65                                           |    |                |
| 9 PHARMACEUTICAL INFORMATION ...............................................................................................67                                       |    |                |
| 9.1.1 CAR-20/19-T CELLS                                                                                                                                              |    |                |
| 9.1.2 Solution Preparation .................................................................................................................68                       |    |                |
| 9.1.3 Investigational Agent Administration .................................................................................68                                       |    |                |
| 9.1.4 Storage Requirements .............................................................................................................68                           |    |                |
| 9.1.5 S TABILITY .                                                                                                                                                   |    |                |
| 9.1.6 Route of Administration ................................................................................................................69                     |    |                |
| 9.1.7 Nursing Implications ..........................................................................................................................70              |    |                |
| 9.1.8 H ANDLING                                                                                                                                                      |    |                |
| 9.1.9 A VAILABILITY                                                                                                                                                  |    |                |
| 9.1.10 Agent Ordering ................................................................................................................................70             |    |                |
| 9.1.11 A GENT A CCOUNTABILITY .......................................................................................................................70              |    |                |
| 9.1.12 Agent Destruction and Return ..................................................................................................70                             |    |                |
| 9.2.1 Tocilizumab ................................................................................................................................................70 |    |                |
| 9.2.2 Preparation                                                                                                                                                    |    |                |
| 9.2.3 T OCILIZUMAB A DMINISTRATION                                                                                                                                   |    |                |
| IT-CAR-T-Shah                                                                                                                                                        | 15 | Version No.: 5 |
| Version Date:6/18/2019                                                                                                                                               |    |                |  
| 9.2.4 S TORAGE                                                                                                                                                 |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 9.2.5 S TABILITY .                                                                                                                                             |
| 9.2.6 H ANDLING                                                                                                                                                |
| 9.2.7 A VAILABILITY                                                                                                                                            |
| 9.2.8 Agent Ordering .                                                                                                                                         |
| 9.2.9 Agent Destruction and Return ........................................................................................................72                  |
| 10 REPORTING AND DOCUMENTING RESPONSE (MEASUREMENT OF EFFECT) ...........72                                                                                    |
| 10.1 EVALUATION OF EFFICACY (OR ACTIVITY)                                                                                                                      |
| 10.2 R ESPONSE C RITERIA .                                                                                                                                     |
| 10.3 CHRONIC LYMPHOCYTIC LEUKEMIA RESPONSE CRITERIA                                                                                                            |
| 10.4 N ON -H ODGKIN ' S L YMPHOMA R ESPONSE C RITERIA ..............................................................................74                         |
| 10.5 R ESPONSE D EFINITIONS ............................................................................................................................75     |
| 10.6 EVALUATION OF SAFETY ...............................................................................................................76                    |
| 1 CORRELATIVE STUDIES/SPECIAL STUDIES                                                                                                                          |
| 1.1 T C ELL D VERSITY A NALYSIS D URING CAR -T C ELL T HERAPY ...................................................76                                            |
| 11.2: Circulating Tryptophan Metabolites and Gene Expression Patterns and risk for                                                                             |
| CNS TOXICITY DURING CAR-T CELL THERAPY .                                                                                                                       |
| 1.3: P ATIENT - REPORTED OUTCOME (PRO) MEASURES FOLLOWING CAR - T CELL THERAPY .......78                                                                       |
| 12 STATISTICAL CONSIDERATIONS                                                                                                                                  |
| 12.1 S TUDY E NDPOINTS ............................................................................................................................80          |
| 12.2 A CCRUAL E STIMATES  .................................................................................................................................80  |
| 12.3 Interim Analyses and Stopping Rules .......................................................................................80                             |
| 12.4 S TATISTICAL A NALYSES P LANS ....................................................................................................80                      |
| 12.5 A NALYSIS P OPULATION .................................................................................................................................82 |
| 13 DATA AND SAFETY MONITORING PLAN (DSMP) ......................................................................82                                             |
| 13.1 S TUDY T EAM                                                                                                                                              |
| 13.2 Quality Assurance ...........................................................................................................................82           |
| 13.3 C LINICAL T RIALS O FFICE                                                                                                                                 |
| 13.4 DSMC                                                                                                                                                      |
| 14 REGULATORY COMPLIANCE, ETHICS AND STUDY MANAGEMENT .............................83                                                                          |
| 14.1 E THICAL S TANDARD                                                                                                                                        |
| 14.2 R EGULATORY C OMPLIANCE                                                                                                                                   |
| 14.3 I NSTITUTIONAL R EVIEW B OARD  .................................................................................................................84        |
| 14.4 S UBJECT C ONFIDENTIALITY AND A CCESS TO S OURCE D OCUMENTS / D ATA  ...............................84                                                    |
| 14.5 Q UALITY A SSURANCE                                                                                                                                       |
| 14.6 CHANGES IN THE PROTOCOL                                                                                                                                   |
| 15 DATA HANDLING AND RECORD KEEPING ................................................................................86                                         |
| 15.1 O VERVIEW .............................................................................................................................................86 |
| 15.2 S TUDY R ECORD R ETENTION ..............................................................................................................86                |
| 15.3 P UBLISHING D ATA  ....................................................................................................................................86 |
| 16 REFERENCES                                                                                                                                                  |
| APPENDIX A: PERFORMANCE STATUS CRITERIA ..............................................................94                                                       |
| APPENDIX B:  NEUROLOGICAL ASSESSMENT .........................................................................95                                               |  
IT-CAR-T-Shah APPENDIX C: 2004 MAS/HLH DIAGNOSTIC CRITIERA ........................................................96 APPENDIX D: SCHEMA FOR TESTING OF CART CELLS FOR FRESH INFUSION OR
CRYOPRESERVATION (FOR LATER INFUSION) IN **PHASE 1 DOSE** ESCALATION/EXPANSION.....................................................................................................97 APPENDIX E: CAR-T CELL TREATMENT GUIDELINES........................................................98  
| APPENDIX F: ASSESSMENT/MANAGEMENT OF CYTOKINE RELEASE SYNDROME AND  NEUROTOXICITY ASSOCIATED WITH CAR-T CELLS [1, 56] 99 APPENDIX G: SUPPORTIVE CARE MEASURES FOR CAR-T CELL TREATMENT 103 APPENDIX H: OPERATING PROCEDURES FOR PATIENTS TREATED WITH CAR-20/19-T  CELLS AT A NON-SPECIFIED DOSE 104 APPENDIX I: QUALITY OF LIFE (QOL) MEASURES 105 APPENDIX J: SPECIMEN COLLECTION AND HANDLING 115   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
LIST OF ABBREVIATIONS  
| AAHRPP   | Association for the Accreditation of Human Research Protection Programs   |
|----------|---------------------------------------------------------------------------|
| AE       | Adverse Event                                                             |
| ALC      | Absolute Lymphocyte Count                                                 |
| ALL      | Acute Lymphoblastic Leukemia                                              |
| ALP      | Alkaline Phosphatase                                                      |
| ANA      | Anti-Nuclear Antibody                                                     |
| ALT      | Alanine Aminotransferase                                                  |
| ANC      | Absolute Neutrophil Count                                                 |
| AST      | Aspartate Aminotransferase                                                |
| ATC      | Anatomical Therapeutic Chemical (Classification System)                   |
| BCW      | Blood Center of Wisconsin                                                 |
| BUN      | Blood Urea Nitrogen                                                       |
| CAR      | Chimeric Antigen Receptor                                                 |
| CBC      | Complete Blood Cell (count)                                               |
| Cl       | Chloride                                                                  |
| CLL      | Chronic Lymphocytic Leukemia                                              |
| Cr       | Creatinine                                                                |
| CR       | Complete Response                                                         |
| CRC      | Clinical Research Coordinator                                             |
| CRF      | Case Report Form                                                          |
| CRP      | C-Reactive Protein                                                        |
| CRS      | Cytokine Release Syndrome                                                 |
| CSF      | Cerebral Spinal Fluid                                                     |
| CT       | Computerized Tomography                                                   |
| CTCAE    | Common Terminology Criteria for Adverse Events                            |  
| CTEP   | Cancer Therapy Evaluation Program                    |
|--------|------------------------------------------------------|
| CTMS   | Clinical Trial Management System                     |
| DFS    | Disease-Free Survival                                |
| DIC    | Disseminated Intravascular Coagulation               |
| DLT    | Dose Limiting Toxicity                               |
| DLBCL  | Diffuse Large B cell Lymphoma                        |
| DSMB   | Data and Safety Monitoring Board                     |
| DSMC   | Data and Safety Monitoring Committee                 |
| DSMP   | Data and Safety Monitoring Plan                      |
| EBV    | Epstein-Barr Virus                                   |
| ECG    | Electrocardiography                                  |
| ECHO   | Echocardiogram                                       |
| ECOG   | Eastern Cooperative Oncology Group                   |
| ESR    | Erythrocyte Sedimentation Rate                       |
| FACT   | Foundation for the Accreditation of Cellular Therapy |
| FCBP   | Female of Childbearing Potential                     |
| FDA    | Food and Drug Administration                         |
| FH     | Froedtert Hospital                                   |
| FT4    | Free Thyroxine                                       |
| GCP    | Good Clinical Practice                               |
| HAMA   | Human Anti-Mouse Antibody                            |
| HBeAg  | Hepatitis B "e" Antigen                              |
| HBV    | Hepatitis B Virus                                    |
| HCT    | Hematocrit                                           |
| HCV    | Hepatitis C Virus                                    |
| HGB    | Hemoglobin                                           |  
| HIV    | Human Immunodeficiency Virus                           |
|--------|--------------------------------------------------------|
| HLH    | Hemophagocytic Lymphohistiocytosis                     |
| ICH    | International Conference on Harmonization              |
| IHC    | Immunohistochemistry                                   |
| IND    | Investigational New Drug Application                   |
| IP     | Investigational Product                                |
| IRB    | Institutional Review Board                             |
| iwCLL  | International Workshop on Chronic Lymphocytic Leukemia |
| IV     | Intravenous                                            |
| IVIG   | Intravenous Immunoglobulin                             |
| K      | Potassium                                              |
| LDH    | Lactate Dehydrogenase                                  |
| LDP    | Lymphodepleting                                        |
| LP     | Lumbar Puncture                                        |
| LPL    | Lymphocyte Propagation Laboratory                      |
| LFT    | Liver Function Test                                    |
| MAS    | Macrophage Activation Syndrome                         |
| MCWCC  | Medical College of Wisconsin Cancer Center             |
| MedDRA | Medical Dictionary for Regulatory Activities           |
| MMSE   | Mini-mental Status Exam                                |
| MNC    | Mononuclear cells                                      |
| MRI    | Magnetic Resonance Imaging                             |
| MUGA   | Multigated Acquisition Scan                            |
| Na     | Sodium                                                 |
| NAAT   | Nucleic Acid Amplification Test                        |
| NCI    | National Cancer Institute                              |  
| NHL    | Non-Hodgkin's Lymphoma                                       |
|--------|--------------------------------------------------------------|
| ORR    | Overall Response Rate                                        |
| PET/CT | Positron Emission Tomography-Computed Tomography             |
| PD     | Progressive Disease                                          |
| PO     | Per os (by mouth, orally)                                    |
| PR     | Partial Response                                             |
| QOL    | Quality of Life                                              |
| Q-PCR  | Quantitative Polymerase Chain Reaction                       |
| RBC    | Red Blood Cell (count)                                       |
| RCL    | Replication Competent Lentivirus                             |
| SAE    | Serious Adverse Event                                        |
| SD     | Stable Disease                                               |
| SLL    | Small Lymphocytic Lymphoma                                   |
| SRC    | Scientific Review Committee                                  |
| SGOT   | Serum Glutamic Oxaloacetic Transaminase                      |
| SGPT   | Serum Glutamic Pyruvic Transaminase                          |
| TSH    | Thyroid Stimulating Hormone                                  |
| ULN    | Upper Limit of Normal                                        |
| UP     | Unanticipated Problem                                        |
| UPIRSO | Unanticipated Problems Involving Risks to Subjects or Others |
| VSV-G  | Vesicular Stomatitis Virus-G                                 |
| WBC    | White Blood Cell (count)                                     |
| WM     | Waldenstroms Macroglobulinemia                               |
figure 1 study schema
1 introduction 1.1 background

CD19 and CD20 are antigens expressed on both healthy B cells and a multitude of B cellderived hematological malignancies including non-Hodgkin's lymphomas (NHL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). An estimated 162,000 new cases of hematological malignancies were expected to be diagnosed in 2015.[2]
CD19 and CD20 are both highly attractive targets for B cell neoplasm immunotherapy. CD19 is expressed by most B cell malignancies including NHL, ALL, CLL, hairy cell leukemia, and a subset of acute myelogenous leukemia.[3] CD19 is a 95kDa glycoprotein present on the B cell surface from early development until differentiation into plasma cells. It is a member of the immunoglobulin superfamily and a component of a cell surface signal transduction complex that regulates signal transduction through the B cell receptor. CD19 expression is restricted to B
lineage cells and is not expressed by pluripotent blood stem cells or on most other normal tissues.[4, 5] Several clinical trials demonstrated effectiveness of CD19 chimeric antigen receptor (CAR) modified T cells in patients with B-cell ALL, CLL/SLL, or B-cell NHL. [6-10]
CD20 is a non-glycosylated 33 to 37 kDa phosphoprotein that is expressed on both normal and malignant B cells. It normally functions as a component of signal transduction in growth regulation of B-lymphocytes. CD20, like CD19, is a cell surface receptor restricted to B cell precursors and mature B cells but is lost following plasma cell differentiation.[11, 12] Monoclonal antibodies against CD20 are effective in treating B cell malignancies. Unlike CD19 CAR, CD20 CAR-T therapies are in early stages of development but have shown significant promise in some clinical trials.[13, 14] The primary advantage of adoptive immunotherapy using CAR-T cells compared to alternative modalities lies in the ability of T cells to expand and persist upon antigen binding of the target.  
[15] Persistence of CAR-T cells in patients with refractory NHL and ALL has correlated with sustained progression free survival likely due to the long-term surveillance these cells provide against recurrent malignancy.[6] However, despite encouraging results, not all patients respond to this treatment and even those who have achieved remission may relapse. An important mechanism of relapse to both targeted monoclonal antibody treatment and CAR-T therapy was shown to be downregulation of the targeted receptor allowing tumor cells to escape destruction.[7, 16] Given the overlap of CD19 and CD20 expression on B cells **(Figure 2)** we speculate that simultaneous targeting of these two separate B cell receptors may result in a more complete B cell ablation and a reduced risk of tumor cell escape. In this Phase 1/1b study we first hope to demonstrate the feasibility of manufacturing CAR-T cells expressing scFv specific for CD20-CD19 (CAR-20/19-T) in a completely closed system using the CliniMACS Prodigy device and then determine the safety of this dual targeted CAR in a first-in-human study of patients with relapsed and refractory B cell malignancies. Secondary outcomes will include response rates, and observed toxicities of the treatment, specifically the development of cytokine release syndrome (CRS)[7], an inflammatory storm that has been seen with previous CAR-T therapies.  
Figure 2: B-cell  
 maturation and markers of differentiation
figure 1 study schema
1.2 investigational agent(s)

The investigational agent in this protocol is the CAR-20/19-T cells. The CAR-T cells will be produced in the Lymphocyte Propagation Laboratory (LPL) under an IND to be submitted to FDA prior to trial initiation. The Cell lines will be produced as follows: A non-mobilized mononuclear cell (MNC) apheresis product (MNC, Apheresis) will be obtained from the patient approximately 4 weeks before infusion. The MNC, Apheresis product will be washed with buffer to remove platelets and plasma, treated with a combination of antibodies to CD4 and CD8 that have been conjugated to paramagnetic particles and enriched for CD4+ and CD8+ T cells.  
Minimally 1.0 x 108 enriched cells will be activated using Miltenyi TransACT reagent, which consists of nanoparticles conjugated to CD3 and CD28. The cells will be incubated for 24 hours at 37oC and 5% CO2 prior to transduction. The activated cells will then be incubated with a lenti-virus vector that will introduce a chimeric antigen receptor that will externally express antibody binding regions to CD19 and CD20, with transmembrane signaling domains from 41BB and CD3ζ **(Figure 3)**. The cells will be further cultured in medium containing interleukin-2, fed, and expanded within the Prodigy culture chamber to numbers suitable for clinical use. After 13-14 days of expansion in culture the cells will be washed and tested for expression of the CD20/CD19 antibody and for function using cytotoxicity and IFN-ϒ production assays in response to B cells prior to infusion. The lines will undergo further testing for sterility, viability, average copy number/cell, VSVg qPCR, and for relative subsets include central memory, effector memory, and regulatory T cells. Processing prior to the final preparation for either immediateadministration or cryopreservation will be performed in a closed and sterile system using GMP grade reagents and materials obtained from Miltenyi Biotec within the ISO 7 environment of the LPL. Copy number and VSVg qPCR will be tested by Lentigen Technology.
figure 1 study schema
closed processing using the clinimacs

The ability to produce CAR-T cell lines suitable for clinical use requires manufacturing processes that are reproducible, scalable and that maintain sterility. Most CAR-T cell trials have involved complex manual processing procedures that include: enrichment and/or depletion of cellular subsets from starting cells, activation and exposure to viral vectors to introduce the chimeric antigen receptors, and cell expansion steps that may require weeks in culture. There have been attempts to automate individual steps, or to close the system.[17] However, the CliniMACS Prodigy device (Miltenyi Biotec) is the first computer-controlled unit that automates each of the required processing steps while in a completely closed system.[17, 18] The Prodigy device has been approved for use in cell separation procedures and for the selection and culture of antigen reactive T cells.[19, 20] We will use the CliniMACS Prodigy for the automated, closed system processing of the CAR-20/19-T cell line to be used in this study.
figure 1 study schema
1.3 preclinical data

A wealth of preclinical studies demonstrates the effectiveness of engineered T cells against CD19 and CD20 positive malignancies. While the construction and the vector (gammaretroviral vectors vs lentiviral vectors) used to create CAR T cells has varied, the incorporation of a costimulatory signaling domain in 2nd generation CARs with either CD28 or 4-1BB (CD137)
increases the potency of these cells.[21, 22] Lentiviral vectors have been favored due to their ability to transduce non-dividing and dividing cells, insert a large genetic segment and allow for long-term transgene expression.[23-25] The pre-clinical data supporting the efficacy of CAR cells in xenograft models is published [26-28] and more recently the feasibility of the production of a dual antigen CAR-20/19-T cell was presented.[29] This preclinical model established the in vitro effectiveness of cytotoxic tumor cell killing by dual expressing CARs and also demonstrated a decreased level of antigen escape by down-regulation when compared to single antigen targeted CAR-T cells.[30]
figure 1 study schema
1.4 mcw preclinical data clinical

The CliniMACS Prodigy device was recently introduced to the North American market as a follow-up to the CliniMACS Cell Separation System. The MCW Cell Processing laboratory has an extensive experience with the CliniMACS Cell Separation systems, having employed them since 2005 for clinical protocols involving graft engineering to deplete and/or enrich specific cellular subsets as detailed below:
MCW's experience with the CliniMACS Cell Separation system indicates that the device produces reproducible and highly purified target populations, rigorous depletions of unwanted populations, and consistent recoveries of >50% of desired cell subsets. The MCW Cell Therapy Lab has additional experience with use of this device in a multi-center trial conducted by the Blood and Marrow Transplant Clinical Trial Network (CTN), CTN0303 that resulted in the FDA approval of the device for clinical use. Dr. Taylor (previous Lab Dierctor) had primary responsibility for preparing the Standard Operating Procedures for the device's use and analyzing the device's performance in that trial [31] as well as her current participation on the steering committee of the ongoing CTN1301 multi-center trial. The Prodigy device incorporates the same technology as the Cell Separation System but with additional functionalities that include pre-separation steps and post-separation culture steps.
figure 1 study schema
preclinical experience using clinimacs prodigy

In September 2015, Dr. Fenlu Zhu, Assistant Lab Director, underwent 2 ½ days of intensive training to learn the steps of CAR-T cell production via the Prodigy device at Lentigen Technology, Inc. Eight preclinical experiments using the CliniMACS Prodigy were conducted. Three experiments were performed using a CD19 vector with a design similar to the CD20/CD19 vector to be used clinically, 4 were using a research grade production of the vector to be used clinically, and the 5th experiment used the GMP grade vector that will be used for the clinical protocol. For all 8 experiments cells were processed through the CD4/CD8 positive cell enrichment phase. There were two failed experiments of these first 8 studies. One failure was due to contamination of the starting cell product caused by a failure of aseptic technique during an open processing step; a step that would not be a factor for the clinical study. The second failure was device-related secondary to a software update that was not properly verified by the service technician from Miltenyi. The error prevented us from moving forward after the cell enrichment phase. The first line that was generated was compromised by the decision to start the experiment even though the source material was 72 hours old due to delays in receiving it into the laboratory. Going forward product processing will begin no longer than 48 hours after collection. The results obtained are summarized below.  
| Cell Population   | % of cells   | % recovery   |
|-------------------|--------------|--------------|
| CD4+ plus CD8+    | 98±4         | 54±7         |
| CD3+              | 90±10        | 54±9         |
| CD3+CD4+          | 65±8         | 60±9         |
| CD3+CD8+          | 26±11        | 52±16        |
| CD3-CD56+         | 6±9          | 27±15        |  
CD4/CD8 Enrichment: Data for enrichment were obtained for all 8 lines and are summarized in the table below. Purity and Recovery post CD4/CD8 enrichment (N=8)
The enriched cells were highly purified for CD4+ and CD8+ cells. While CD4 is limited to T
cells, CD8 is also expressed on a subset of NK cells that may or may not be CD3+. An average of 90±10% of the enriched cells were CD3+ and 6±9% were CD3-CD56+ NK cells that express a form of CD8 that has a lower antigen density than CD3+CD8+ T cells. >50% of the starting target population of CD4+ plus CD8+ cells were recovered. Importantly monocytes, which are known to suppress T cell proliferation of patient-derived material, were absent.[32, 33]
Cell Expansion: Cell lines were started using 1.0 x 108 starting CD4/CD8 enriched cells and were harvested on day 13. The first experiment using a buffy coat product as the starting material expanded only 5.4-fold from the start, likely due to the age of the cells at the start of processing. One line using the CD19 vector expanded 25.7-fold and the 4 lines using the CD20/CD19 vector expanded 414.5, 33.8, 24.5 and 24.8-fold. Therefore, expansion exceeded the expected results from the Lentigen training experience of approximately 20-fold Transduction efficiency: The percentage of viable transduced cells was measured by Protein L
expression, a protein unique to immunoglobulin kappa light chains.[34] Expression is shown in the table below and flow data from one representative line is shown in **Figure 4**.  
| Vector (MOI)      | % of CD3   | % of CD4   | % of CD8   | Copy#/All Cells   |
|-------------------|------------|------------|------------|-------------------|
| #1 CD19 (5)       | 41.0       | 42.3       | 26.4       | 1.01              |
| #2 CD19 (5)       | 45.7       | 52.3       | 43.7       | 0.48              |
| #3 CD20/CD19 (5)  | 21.0       | 22.7       | 16.5       | 0.23              |
| #4 CD20/CD19 (10) | 34.0       | 28.7       | 35.4       | 0.53              |
| #5 CD20/CD19 (10) | 56.6       | 57.6       | 60.3       | 0.67              |
| #6 CD20/CD19 (10) | 25.8       | 28.7       | 23.9       | nd                |  
Transduction efficiency as measured by Protein L expression  
  
Post Expansion Phenotype: The phenotype of the expanded population changed from the starting CD4/CD8 enriched cells in that fewer CD3-CD56+ NK cells expanded, as shown most clearly for line \#3. The percentage of CD8+ cells increased from the starting cells expect for Line \#1 where the line was greatly enriched for CD4+ cells, possibly due to the age of the cells at the start of the culture. The goal is to have both CD4+ and CD8+ cells in the cultures, though cytotoxicity is expected to be mediated predominately by the CD8+ T cells. CD4+ CAR cells are expected to provide a helper function to the CD8+ cells secondarily to activation after exposure to the ligand bearing B cell. CAR-T cell line viability at the time of harvest was 90.1± 6.5%.  
| Line Designation   | %CD3+   | %CD3+   | %CD3+   | %CD3-   |       |
|--------------------|---------|---------|---------|---------|-------|
| %CD3               | CD4+    | CD8+    | CD56+   | CD56+   |       |
| Line#1 Pre         | 99.5%   | 74.8%   | 23.9%   | 0.9%    | 0.2%  |
| Post               | 99.3%   | 93.8%   | 8.5%    | ND      | 0.6%  |
| Line #2 Pre        | 97.9%   | 51.7%   | 49.5%   | 4.2%    | 2.2%  |
| Post               | 98.8%   | 29.3%   | 71.8%   | 4.3%    | 1.1%  |
| Line #3 Pre        | 78.7%   | 63.2%   | 16.2%   | 1.7%    | 18.4% |
| Post               | 99.5%   | 50.2%   | 47.1%   | 1.6%    | 0.5%  |
| Line #4 Pre        | 91.7%   | 64.9%   | 27.9%   | 8.9%    | 1.1%  |
| Post               | 97.3%   | 46.5%   | 50.5%   | 9.7%    | 0.5%  |
| Line #5 Pre        | 90.1%   | 75.0%   | 16.3%   | 2.4%    | 0.6%  |
| Post               | 98.5%   | 42.7%   | 59.1%   | 3.4%    | 0.9%  |
| Line #6 Pre        | 90.3%   | 69.4%   | 22.5%   | 2.3%    | 1.7%  |
| Post               | 99.4%   | 58.3%   | 43.7%   | 1.4%    | 0.1%  |  
It is important that any method of CAR-T cell production should preserve a population of central memory T cells to ensure longevity in circulation. An advantage of immunotherapy using CART cells is that during transduction the cells are not being expanded through antigen stimulation and thus should not become enriched for T cells with an exhausted phenotype (Terminal Memory T cells). As shown in **figure 5** below for Line\#5, a population of Central Memory CD4+
and CD8+ T cells are maintained using the CAR-20/19-T cell vector manufactured both in the CliniMACS Prodigy and using plates followed by expansion in the G-Rex Device (Wilson-Wolf, Inc).
figure 1 study schema
figure 5 t cell subset

Lines \#1, \#2, and \#3 were tested for the presence of regulatory T cells (Tregs) before and after line generation. Low numbers of Tregs were present in the starting fractions, but were undetectable in the lines post expansion, indicating that our approach does not enrich for populations that might suppress tumor killing.  
Effector cell function: Effector cell function was measured both in 51Cr release cytotoxicity assays and by assessment of intracellular gamma interferon production after target cell coculture. All the lines, except the Line \#1 experiment, mediated lytic activity to B cell lines expressing both CD19 and CD2 (Raji or EBV transformed B cells). This was likely due to the failure of the CD8+ transduced cells to expand in this line. However, both the few CD8+ cells and the CD4+ cells produced IFN-γ after co-culture with B cell targets We obtained K562 cells that were separately transduced with CD19 and CD20 after production of Line \#4 the second CD20/CD19 transduced line and could demonstrate preferable killing of both transduced targets in addition to Raji cell killing, though this line did contain a higher percentage of CD3+CD56+ cells than the other lines after expansion that resulted in detectable lysis of the parental K562 line. However, an IFN-ϒ intracellular cytokine assay showed cell activation only after co-culture with the transduced lines, Raji cells, but not parental K562 as shown in Figure 6.  
Similar results were obtained with Line \#5 where there was lysis of the parental K562 line that was greatly reduced by depletion of CD56+ cells, but failed to stimulate IFN-Y production even without CD56+ cell reduction.  For both Line \#4 and Line \#5 depletion of CD56+ cells had little effect on the killing of CD19 or CD20 expressing target cells.
figure 1 study schema
figure 6 effector function of

Figure 7: Effect of  
  
CD56+ cell depletion on lysis of parental K562 targets
figure 1 study schema
1.5 clinical data b-cell all

ALL is a fatal lymphoid malignancy that can develop in patients of all ages. Although children with ALL have impressive outcomes with combination chemotherapy, those with high-risk cytogenetics or a Philadelphia (Ph) chromosome do not fare as well despite intensive therapy and allogeneic transplantation. Adults have significantly worse prognosis than children.[35]
Therapies for relapsed or refractory ALL are limited, and many patients are unable to tolerate the successive lines of cytotoxic chemotherapy necessary to achieve disease control.[36]
CD19 positivity is almost ubiquitous in ALL and is a marker used in diagnosis of the disease.  
Consequently, many of the first CD19 CAR-T cell studies involved patients with either relapsed or refractory ALL with remarkable outcomes. In a combined pediatric/adult ALL study, a complete remission (CR) was achieved in 90% of patients with relapsed ALL with sustained remissions in two-thirds of those patients.[6]
figure 1 study schema
b-cell lymphomas

There are several subtypes of B cell NHL, including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), CLL/SLL (small lymphocytic lymphoma), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and lymphoplasmacytic lymphoma (LPL)/Waldenstroms Macroglobulinemia (WM). As these are mature B cell malignancies, CD20 expression is almost universal, and treatment with rituximab is standard of care in all subtypes.[37] Standard first line therapy includes the CD20 antibody rituximab in combination with cytotoxic chemotherapy. After relapse, salvage chemotherapy +/- autologous transplant are considerations and can cure a proportion of patients. However, treatment options for those failing second line treatment or refractory to first or second line treatment are quite limited.[38] Similar to CD20, CD19 expression is also seen in most B cell lymphomas although the level of expression varies with histologic subtype.[39] CD19 CAR-T therapy resulted in dramatic responses in a heavily pretreated population of patients with multiple histologic subtypes of lymphoma. The initial publication for CAR-T cell therapy against CD19 demonstrated significant efficacy in 3 patients with relapsed, refractory CLL[40]. In a single arm study utilizing a CD28, CD3ζ co-stimulatory domain CAR-T cells, 8/15 patients treated with anti-CD19 CARs achieved a CR. Four of seven patients with chemo refractory DLBCL achieved CR; these were durable in 3 patients.[9] In another study, presented at ASH 2015, patients with relapsed, refractory DLBCL, MCL, and FL treated with CD19 CAR-T cells had an overall response rate of 68% with minimal toxicity.[10] More recent data from ASH 2016 from KITE, a large pharmaceutical company evaluating CD19 CAR-T cells, demonstrated an overall response rate of 76% in patients with relapsed, refractory NHL with 39% of patients having an ongoing response at 3 months.[41] Additionally, from ASH 2016, ibrutinib refractory patients receiving CAR-T cells against CD19 were found to have an overall response rate of 76% with CRs achieved in 5 patients.[42] These above data support the efficacy of CAR-T cells in both NHL and CLL.
figure 1 study schema
car-t therapy

The CAR approach uses genetically programmed, patient-derived lymphocytes transfected with chimeric receptor genes to combine the effector functions of T lymphocytes with the ability of antibodies to recognize predefined surface antigens with high specificity in a non-major histocompatibility complex (MHC) restricted manner.[43] Consequently, CAR-T cells have the ability to recognize intact membrane proteins independent of antigen processing or presentation by the host's immune cells. CARs typically encode a single chain variable fragment (scFv) extracellular domain to bind tumor or virus linked to an intracellular signaling domain that mediates T cell activation. In principle, universal targeting vectors can be constructed because the scFv binds to native cell surface epitopes and bypass the requirement for MHC restriction (interaction with recipient HLA molecules on host immune cells). The scFv antibody, contains the VH and VL chains joined by a peptide linker of about 15 residues in length.[44] We intend to use a second generation CAR approach which provides costimulatory signaling domains 4-1BB
and CD3ζ together with *two scFv* chains recognizing both CD19 and CD20.[15, 45] **(Figure 3)**
There are several potential limitations to activity of CAR-T cells: 1) the tumor must express the target antigen on the cell surface; 2) large amounts of shed or soluble antigen could inhibit the CAR-T cells; 3) the chimeric receptor may be immunogenic, resulting in the elimination of the redirected T cells by the host immune system.
figure 1 study schema
1.6 rationale for lymphodepletion and

It is increasingly clear that adoptive immunotherapy strategies are more effective in the context of homeostatic T cell proliferation - the proliferation of naive T cells and their differentiation into memory-like T cells when total numbers of naive T cells are reduced by lymphodepleting chemotherapy. Lymphodepletion also eliminates regulatory T cells and other competing elements of the immune system that act as "cytokine sinks", enhancing the availability of cytokines such as IL-7 and IL-15.[46, 47] In this protocol we propose to transfer CAR-20/19-T cells into subjects that are rendered lymphopenic as a result of cytotoxic chemotherapy.  
The optimal regimen for lymphodepletion remains unknown. Various CAR trials have utilized a combination of fludarabine, cyclophosphamide, pentostatin, or disease specific chemotherapeutic agents.[7, 9, 10, 40, 48, 49] Given the heterogeneity of previous CAR trials and using the previously published literature, we will, like most other institutions, utilize a combination of a purine analog (fludarabine) with cyclophosphamide, which is the most common lymphodepletion regimen used.[48, 49] The regimens for this trial is listed in section 6.1.
figure 1 study schema
dose rationale

Previous literature has demonstrated a response to CAR-T cells at a variety of dose levels from 1.0-5.0 x106 cells/kg to 1.0-5.0x109 CAR-T cells as a flat, weight independent dose.[9, 10, 40]
Although an optimal dose has not been determined, based on prior studies, our goal dose level is 2.5 x 106 cells/kg. However, due to concern for increased toxicity with a dual targeted CAR-T
cell, we will start at a dose level of 2.5 x 105 cells/kg and dose escalate per our study design
(Section 6.4) to our target dose. Based on FDA recommendations we will dose per kg for uniformity and the dose will be maxed at a weight of 80 kilograms. Recent data regarding cell dose optimization presented at the American Society of Clinical Oncology meeting in 2016 demonstrated that administration in split-dose fashion may improve safety without altering efficacy and that a flat cell dose of 108 was the optimal dose level. Higher doses led to more CRS related deaths. [50, 51] Based on that data we will split our infusion during the phase 1 portion over 2 days, 30% on Day 0 and 70% on Day 1. If there is no significant toxicity during the phase 1 portion, we will transition to single day dosing in our Phase 1b portion of the study. Phase 1b will be open to enrollment once a subject has consented for the final slot on the Phase 1 portion. Our target dose was chosen on the basis of efficacy and safety data from CD19 directed CAR-T cells with similar 4-1BB and CD3ζ co-stimulatory domains.[48, 49] Given that CAR-T cell expansion occurs in vivo post infusion, there has not been clear evidence of dose response related to efficacy or toxicity.
figure 1 study schema
1.7 known and potential risks

Patients enrolled on this study will be exposed to a genetically engineered autologous T cell. Potential risks of the CAR-20/19-T cells include the following:
- B cell *ablation and consequent increased risk of infection*: As both healthy and malignant B cells express CD19 and CD20 antigens, B cell ablation with consequent elimination of immunoglobulin production is an expected side effect of administration of the CAR20/19-T cells. This may be transient or permanent depending on the longevity of the CAR-T cells; it generally resolves when the CAR-20/19-T cells are cleared. Patients with persistent low levels of immunoglobulins can be given intravenous immune globulin if felt to be indicated by their treating physician.  
- *Immune Reaction*: Although we are utilizing autologous T cells, it is possible that the processing will make these cells immunogenic and that patients will have an immune response directed against the scFv. If such a response occurs, it is possible that the cells will be rejected or a serious allergic reaction will occur. Thus far, such allergic reactions have not been reported in clinical studies of CAR-T cells. [6, 8, 9, 40] Additionally patients can get exacerbation of pre-existing auto-immune conditions or development of new auto-immune or neurologic conditions.  
- *Transformation:* Gene transfer may result in new tumors derived from genetically modified cells, due to incorporation of viral DNA, which may dysregulate genes controlling cell apoptosis and proliferation leading to an uncontrolled malignant clone or new hematologic malignancy. Again, this is not yet reported in clinical studies of CAR-T
cells in human patients, which now number greater than 100. [6, 8, 9, 40]
- Graft-Versus-Host Disease *(GVHD)*: In patients who have underwent previous allogeneic transplant and then relapse with disease and receive CAR-T therapy with allogeneic cells, one potential risk is re-activation of GVHD. This was not seen in one recent prospective study of patients receiving allogeneic CAR-T cells from their donor. [52]
- Risk of tumor lysis syndrome (TLS) related to lymphodepleting chemotherapy or CAR-T
cells: The risk of TLS is dependent on the burden of disease and rapidity of response.  
There are reports of patients treated with CD19 CAR-T cells developing tumor lysis syndrome. Therefore, all patients will be closely monitored both before and after
chemotherapy and CAR-20/19-T cell infusions including blood tests for potassium, uric acid, and calcium. All subjects will receive allopurinol prophylactically. TLS resulting in renal insufficiency, or rapidly rising uric acid, or evidence of organ dysfunction will be managed with intravenous fluids and rasburicase as needed and determined by the treating physicians. Appropriate clinical therapy will be administered should any significant TLS occur.  
- *Cytokine Release Syndrome (CRS)*: CRS is an inflammatory syndrome that occurs as a result of T cell activation and proliferation associated with engagement of the targeted antigen. It manifests when large number of lymphocytes become activated and release inflammatory cytokines. Its symptoms (**Figure 8**) can vary from mild flu-like complaints with fevers, anorexia, nausea/vomiting, headache, and rash to a severe inflammatory storm that can result in hypotension, multi-system organ failure, shock, and even death despite treatment with anti-cytokine therapy and steroids. [1, 53, 54] It is associated with markedly increased production of acute phase reactants such as C-reactive protein
(CRP) and ferritin as well as increases in the erythrocyte sedimentation rate (ESR). The presentation of CRS is most similar with MAS (macrophage activation syndrome) or HLH (hemophagocytic lymphohistiocytosis) and is felt to be a reaction to the immune activation that occurs with antigen binding by the CAR-T cells. Diagnosis of MAS or HLH
is based on the 2004 HLH criteria (Appendix C). [55] It is unclear if the development of CRS predicts clinical response. Initial reports of CRS
in CAR-T cell clinical trials revealed dramatically increased levels of IL-6 that peak during maximal T cell proliferation. Inhibition of IL-6 binding with the IL-6 receptor antibody, tocilizumab results in a rapid and dramatic improvement in the life-threatening symptoms associated with CAR-T therapies. It is unclear if this inhibition dampens the efficacy of the modified T cells.[53, 56] Given the unique presentation of CRS
associated with CAR-T cell therapy, a revised grading system has been recommended for appropriate classification of degree of toxicity. We will employ this revised grading system when evaluating patients with CRS in this study.[1]  
  
  
- *CRS related Neurological Toxicity:* Neurological toxicity is a specific risk that has been observed with anti-CD19 CAR-T cell therapy. This has manifested with symptoms such as hallucinations, delirium, aphasia, and confusion. These toxicities range from mild to severe (requiring ventilator support for airway protection).[1, 53, 57, 58] Neurological toxicity generally emerges within the first week after anti-CD19 CAR-T cell infusion, and resolves within a few days. Similar neurological changes are observed with blinatumomab administration and CD28 mAb ligation, for which it has been speculated that neurological toxicity arises from generalized T cell mediated inflammation rather than direct toxicity mediated by T cells on CNS tissue.[56] The neurological toxicities reported with anti-CD19 CAR-T cell therapy to date have generally been medically manageable and reversible. However, recently Juno Therapeutics reported 3 deaths due to CNS toxicity as a result of their anti-CD19 CAR-T cell trial.[59] Their CAR utilizes a different signaling domain than the one proposed in this study. In addition, we will require prophylactic levetiracetam in all patients at the time of lymphodepletion chemotherapy to prevent potential neurotoxicity.  
- *Infection*: Patients who receive fresh CAR-T cells are at risk of developing an infection as results of complete sterility testing will not be available until after infusion. To decrease this risk, in process testing (Appendix D) will be performed prior to start of lymphodepletion chemotherapy. Should the cultures return positive post-infusion appropriate antibiotics and infectious disease work-up will be performed.
figure 1 study schema
potential risks associated with lymphodepleting

A combination of fludarabine with cyclophosphamide will be utilized for lymphodepletion in all patients undergoing treatment with CAR-20/19-T cells. Potential risks associated with this combination chemotherapy include the following
- *Cytopenias*: Cytotoxic chemotherapy can result in drug-induced pancytopenia placing the patient at risks of several complications including bleeding related to thrombocytopenia, infection related to neutropenia, or fatigue/hypotension associated with anemia. Patients will be allowed to receive prophylactic transfusions as indicated in Appendix E. In the event of neutropenic fever, patients will be appropriately managed with broad spectrum antibiotics.  
- *Tumor Lysis Syndrome:* As defined above, patients will be monitored for TLS post treatment with both lymphodepleting and CAR-20/19-T cell treatment.  
- *Organ Toxicity:* Any cytotoxic chemotherapy regimen can result in organ damage as defined as hypoxia (requiring O2), an elevated creatinine, neurological toxicity, or elevations in liver function tests. These parameters will be monitored per our follow-up schedule in Table 1.
figure 1 study schema
unknown risks

In the setting of genetic therapy and a first-in-human study there are potential unknown risks. The degree of CRS with dual antigen binding has not been previously tested; it is possible that this will lead to a more robust immune response, and therefore more severe CRS, than previously seen. Additionally, as there is limited long-term data in patients treated with CAR-T cell therapies, it is unknown if there will be treatment-related secondary malignancies or other unexpected toxicities in the future.
figure 1 study schema
potential benefits

Patients with relapsed leukemia or lymphoma as based on the inclusion criteria have very poor outcomes with standard treatments options. Allogeneic transplant can be offered to those patients in remission but this comes with high treatment related mortality and many patients are not eligible due to active or progressive disease. Recent data published and presented at national conferences establishes the efficacy of CAR-T cells in the treatment of relapsed, refractory leukemia and lymphoma [6, 8-10, 40] Although clinical responses are plausible, given this is a phase 1/1b study the clinical benefit of this specific CAR-20/19-T cell infusion is unknown.
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2 hypothesis and objectives 2.1

Single antigen targeted monoclonal antibodies and CAR-T therapies have significant efficacy in the treatment of patients with lymphoma and leukemia. However, not all patients respond, and those that do respond can still experience progressive disease. In the setting of monoclonal antibodies such as rituximab (CD20 targeted antibody), one method of resistance has been the downregulation of the CD20 receptor leading to tumor cell escape.[16] Similarly, the proposed mechanism of relapse in CD19 positive ALL treated with CD19 CAR-T cells has been downregulation of the CD19 receptor and development of a clonal CD19 negative population.[7]
Preclinical studies have demonstrated the decreased downregulation of tumor cell antigens when treated with combined CD20/CD19 CAR-T cells.[29] We hypothesize that a dual targeted CD20/CD19 CAR-T cell treatment will be more efficacious than single antigen targeting and will decreased the risk of progression or relapse after treatment.
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2.2 primary objectives

1. Determine the safety (dose level) of CAR-20/19-T cell administration in relapsed refractory CLL/SLL and B-cell NHL
2. Determine the feasibility of manufacturing CAR-20/19-T cells locally from patient apheresis products using the CliniMACS Prodigy Cell processing device
figure 1 study schema
2.3 secondary objectives

1. Determine the anti-tumor responses as measured by response rates.  
2. Describe the duration of response for responding patients 3. Determine persistence of CAR-20/19-T cells 4. Determine the effects of CAR-20/19-T infusion on non-neoplastic CD19 & CD20 B cells in vivo 5. Determine if cellular or humoral host immunity develops *against* CAR-20/19-T cells 6. Evaluate MRD using molecular technologies
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2.4 rationale for the outcome

As a first-in-human and Phase 1 study, our primary outcome is to demonstrate both the safety and feasibility of CAR-20/19-T cell production and administration to patients. Production feasibility will be measured by our ability to perform apheresis and produce gene modified CAR20/19-T cells that can be administered to enrolled patients. Safety will be determined by assessments of CRS using the Lee et al. grading scale [1] and adverse events via NCI CTCAE version 5.0. Response assessment will vary by specific disease subtype and will occur initially at Day +28 post-treatment (+/- 3 days) with CAR-20/19-T cells. Ongoing response assessment will continue until progression and/or death up to 2 years (see Table 1), after which patients will be transitioned to a long-term follow-up protocol.  
At a minimum, the Day +28 (+/- 3 days) response assessment will include the following: Bone Marrow Aspirate and Biopsy and either PET/CT or CT neck/chest/abdomen/pelvis
figure 1 study schema
3 study design 3.1 general

This is a phase 1/1b, interventional single arm, open label, treatment study designed to evaluate the safety and feasibility of infusion of CAR-20/19-T cells in adult patients with B cell malignancies that have failed prior therapies. The primary objective is safety of infusion and feasibility of manufacturing CAR-20/19-T cells at MCW-FH. We will monitor and describe safety, tolerability and engraftment potential of CAR-20/19-T cells in these patients. The general protocol schema is shown in **Figure 1**.  
Subjects who have adequate T cells as per eligibility criteria will be leukopheresed to obtain a MNC apheresis product for CAR-20/19-T manufacturing. The apheresis product 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 CD19 and CD20 scFv regions coupled to CD3ζ/4-1BB signaling domains using a lentiviral vector. The cells will be expanded *in vitro* washed from culture, removed from the device, and either administered to the patient within 48 hours of production or cryopreserved for later administration. In process and end of production release testing will be performed (**Appendix D** for schema) The entire manufacturing process up until removal from culture will be performed using a computer controlled program within the closed system of the CliniMACS Prodigy device. Patients will receive CAR-20/19-T cells through a central venous catheter and then will be monitored closely with physical examinations and blood tests to assess safety, engraftment, and persistence of the CAR-20/19-T cells at regular intervals as outlined (Table 1). Response assessments will also be performed for secondary endpoint analysis as described in Section 3.5. Patients will be followed daily from Day 0 to Day +7. Then seen on Day +10 (+/- 1 day), Day +14 (+/- 2 days), Day +21 (+/- 2 days) and then on Day 28 (+/- 3 days). After Day +28 patients will be seen monthly up to 6 months, every 3 months up to year 1, and every 6 months up to year 2. Details of further follow-up are in listed in the study calendar. Following completion of this study, subjects will be enrolled onto a roll-over long-term follow-up protocol for up to an additional thirteen years to assess the diagnosis of long-term health problems, such as development of new malignancies and persistent of CAR-T cells.
figure 1 study schema
3.2 number of subjects

A maximum of 24 patients will be treated with CAR-20/19-T cells. In the phase 1 portion an initial dose escalation cohort using 3+3 patients at the first dose level in this study. Once a safe dose level has been identified, there will be a 6 patient dose expansion cohort at the selected dose level. Once the final subject has enrolled on the Phase 1 portion of the study, the Phase 1b portion of the study will be open to accrual of 9 patients).
figure 1 study schema
3.3 primary endpoint(s) primary feasibility

Ability to manufacture CAR-20/19-T cells from patient apheresis products using the CliniMACS Prodigy processing device (Miltenyi Biotec). The number of T cell lines that do not meet criteria for T cell purity, total transduced T cell content, viability, and sterility will be determined.
figure 1 study schema
primary safety endpoints assessed include

Occurrence of adverse events, defined as either CRS related Grade 3/4 toxicity or other NCI CTCAE version 5.0 non-hematologic ≥ grade 3 signs/symptoms, laboratory toxicities and clinical events that are possibly, probably or definitely related to study treatment at any time from the infusion until week 4. The revised CRS grading system will be used for patients who develop CRS **(Appendix G)**.[1] This will include infusion toxicity and any toxicity possibly related to the CAR-20/19-T cells including but not limited to:
a. Fevers b. Rash c. Hepatic dysfunction d. Pulmonary infiltrates or other pulmonary toxicity (unrelated to concomitant infections)
e. CRS/MAS
f. Neurologic toxicity
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3.4 dose limiting toxicity

Dose-limiting toxicity (DLT) will be defined as a grade 3-4 non-hematologic toxicity possibly, probably, or definitely related to the infusion of CAR-20/19-T cells. All deaths (grade 5 toxicity)
in the first 28 days felt to be either possibly, probably, or definitely related to CAR-20/19-T cell therapy will be considered a DLT. All Grade 4 non-hematologic toxicity felt to be either possibly, probably, or definitely related to CAR-20/19-T cell infusion will be considered a DLT. If multiple toxicities are seen, the presence of a DLT will be based on the most severe toxicity experienced. The DLT will be based on the tolerability observed during the first 28 days of treatment/observation. CRS and neurotoxicity are possible DLTs that can occur as a result of administration of CAR-T cells. For this protocol, any grade 4 CRS related toxicity per revised CRS grading system
(Appendix G) will be considered a DLT. In addition, patients with Grade 3 CRS toxicity that does not improve to grade 1 or 2 despite treatment with cytokine inhibition and/or immunosuppression within 7 days will also be considered a DLT. All Grade 4 neurotoxicity will also be considered a DLT and similarly any Grade 3 neurotoxicity which does not improve to grade 1 or 2 despite treatment within 7 days will also be considered a DLT. All infusional reactions ≥grade 2 lasting more than 24 hours will be considered a DLT. Lastly, while hematologic toxicity is an expected side effect of lymphodepletion, persistent Grade≥3 neutropenia that does not resolve to Grade≤2 neutropenia by Day +28 will be considered a DLT. Refractory thrombocytopenia and anemia are known potential complications among heavily pre-treated patients with hematological malignancies and lymphodepletion chemotherapy and will be managed per supportive care guidelines. While grade and toxicity will be recorded for this clinical trial the degree of thrombocytopenia/anemia will not count towards our DLT evaluation.  
The first 28 days were chosen for the DLT monitoring period given the variability in the presentation of CRS, which can occur within hours to weeks after infusion [1]. Additionally, as more severe CRS tends to occur earlier after infusion compared to non-severe CRS, 28 days was felt to be an appropriate monitoring period.[6] The attribution of adverse events with respect to their relationship to the infusion of CAR-20/19-T cells will be the responsibility of the site PI.
figure 1 study schema
3.5 secondary endpoint(s) secondary endpoints

1. The number of patients who have CAR-20/19-T cells manufactured but not infused due to failure to meet release criteria.  
2. Disease assessment will include the following:
o Number of patients with anti-tumor responses after CAR-20/19-T cell infusion.  
Since patients with a variety of histologies will be treated - response will be assessed using disease appropriate imaging/marrow and international consensus criteria most applicable to the disease (Section 10).  
o Repeat bone marrow biopsy and aspirate with evaluation of minimal residual disease *when indicated* (i.e., patients with baseline disease on bone marrow biopsy). These will be analyzed by multiparameter flow cytometry (MFC) and/or molecular analysis of immunoglobulin rearrangements, as available.  
o Duration of response in responders o Relapse rates and time to relapse o Cause of death in treated patients o Overall survival and progression free survival of individual patients
figure 1 study schema
exploratory endpoints

1. Characterize CAR-20/19-T cells with respect to their expansion, persistence, phenotype, and function. This will be assessed by flow cytometry, Q-PCR, RNA sequence, and in vitro functional assays.  
2. Determine if host immunity develops against the CAR-20/19-T cell construct or other elements of the transgene or vector and assess correlation with loss of detectable CAR20/19-T cells (loss of engraftment).  
3. Evaluate the effect of CAR-20/19-T cells on B cell and plasma cell compartments assessed by MFC to measure both benign and malignant B cell populations and plasma cell populations.  
4. Describe the incidence of new-onset secondary malignancies if any.  
5. Determine the utility of a T-cell diversity assay in predicting outcomes with CAR-T cell therapy
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3.6 study timeline

Preclinical lines of CD19 and CD20/CD19 CAR T-cells have been generated in support of the IND application. These studies have demonstrated that the MCW lymphocyte propagation laboratory can produce CAR-T cell lines using the Miltenyi CliniMACS Prodigy device. 1) Submission of IND (1-month review time for FDA) 2) Submission of clinical protocol through IRB, DSMC at local institution 3) Clinical accrual after FDA and IRB approved protocol along with regulatory approval from local institutions (MCW and FH).
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3.7 primary completion

The study is expected to reach primary completion 18-24 months from the time the study opens to accrual.
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3.8 study completion

Initial data on the safety, feasibility and short-term toxicity of CAR-20/19-T cells will be complete and available 4 months after completion of accrual. Thus, preliminary toxicity and efficacy data will reach completion 24 months from the time the study opens to accrual.
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4 patient selection 4.1 eligibility

Patients must meet all the below inclusion and exclusion criteria prior to apheresis. After consent patients can receive additional bridging chemotherapy except during listed time periods in inclusion/exclusion criteria prior to apheresis and prior to CAR-20/19-T cell infusion.
figure 1 study schema
4.2 inclusion criteria

1. Diagnosis of B-cell NHL or CLL/SLL: Patients must be aged≥18 years with relapsed, refractory disease and no available curative options that meet clinical criteria to initiate treatment.  
2. Patients with B-cell NHL or CLL/SLL must have either CD19 or CD20 positive disease on most recent biopsy performed (a repeat biopsy is not mandatory for this study except as noted below). A minimum of 5% CD19 or CD20 positivity by immunohistochemistry or flow cytometry on prior or repeat biopsy is required.  
3. Absolute CD3+ T cell count ≥50/mm3 4. MRI brain and Lumbar Puncture with CSF analysis by cytology and flow cytometry without evidence of CNS involvement ONLY in patients with history of CNS involvement 5. Measurable disease must have been documented within 4 weeks of the time of consent defined as the following by disease specific subtype:
c. B-cell NHL: Active disease defined as nodal lesions greater than 20 mm in the long axis or extranodal lesions >10 mm in long and short axis or bone marrow involvement that is biopsy proven d. CLL/SLL: Active disease by either bone marrow, peripheral flow cytometry, or CT
and/or PET imaging with nodal disease 6. Patients should have failed at least two lines of a standard treatment and meet disease specific criteria detailed below:
a. CLL/SLL: measurable disease as defined above that has relapsed after at least one line of chemo-immunotherapy and progressed or intolerant to ibrutinib monotherapy b. CD19 or CD20 positive B cell NHL limited to the following histologies: Advanced Stage III or IV Follicular Lymphoma, Diffuse Large B cell Lymphoma and associated subtypes (e.g. aggressive B-cell lymphoma, T-cell/histocyte rich Bcell lymphoma, primary mediastinal B-cell lymphoma, EBV+ diffuse large B-cell lymphoma, transformed lymphoma such as transformed follicular or marginal zone lymphoma, and Richter's transformation) and Mantle cell lymphoma.  
Specific criteria include:
- Patients must have active, measurable disease after two lines of cytotoxic chemotherapy of which one must be anthracycline containing.  
- Must have received Rituximab or another CD20 antibody and at minimum two chemotherapy regimens appropriate for their disease.  
- Either failed autologous transplant or ineligible to receive autologous transplant 7. Karnofsky performance score ≥70. See Appendix A for scales. 8. Normal Baseline Neurological Evaluation: Mini-Mental Status Exam Score 24-30
(Appendix B)
9. Adequate hepatic function, defined as AST and ALT <5 x upper limit of normal (ULN);
serum bilirubin and alkaline phosphatase <5 x ULN, or considered not clinically significant as per the clinical PIs discretion (e.g. Gilbert's or indirect hyperbilirubinemia) or felt to be due to underlying disease.  
10. Adequate renal function, defined as creatinine clearance>60 ml/min 11. Able to provide written informed consent 12. Agree to practice birth control during the study 13. Adequate cardiac function as indicated by New York Heart Association (NYHA)
classification I or II AND left ventricular ejection fraction of ≥35% (by cardiac ECHO or MUGA) and adequate pulmonary function as indicated by room air oxygen saturation of ≥92%.  
14. Expected survival >12 weeks 15. Negative urine or serum pregnancy test in females of child bearing potential at study entry and again within 48 hours' prior lymphodepleting chemotherapy.  
16. Patients with prior blinatumomab treatment require repeat biopsy post-blinatumomab treatment that demonstrates CD19 or CD20 positive disease.  
17. Meet criteria for regarding fertility and contraception detailed in section 4.4 18. Central line access will be required for CAR-20/19-T cell infusion.
figure 1 study schema
4.3 exclusion criteria

A potential subject who meets any of the following exclusion criteria is ineligible to participate in the study.  
1. Positive beta-HCG in female of child-bearing potential defined as per table 1.  
2. Patients with known systemic allergy to bovine or murine products.  
3. Known prior positive serology for human anti-mouse antibody (HAMA).  
4. Confirmed active human immunodeficiency virus (HIV), Hepatitis B or C infection.  
5. History of significant autoimmune disease OR active, uncontrolled autoimmune phenomenon: such as systemic lupus erythematous, Wegner's glomerulonephritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (AIHA, ITP) requiring steroid therapy defined as >20 mg of prednisone or equivalent daily.  
6. Presence of ≥grade 3 non-hematologic toxicities as per CTCAE version 5.0 from any previous treatment unless it is felt to be due to underlying disease.  
7. Concurrent use of investigational therapeutic agents or enrollment on another therapeutic clinical trial at any institution. Minimum of ≥4 weeks required from administration of any other investigational agents on other clinical trials prior to enrollment on this CAR-T protocol.  
8. Refusal to participate in the long-term follow-up protocol 9. Patients with active CNS involvement by malignancy on MRI or by lumbar puncture.  
a. Patients with prior CNS disease that has been effectively treated will be eligible providing treatment was >4 weeks before enrollment and a remission documented within 8 weeks of planned CAR-T cell infusion by MRI brain and CSF analysis.  
10. Previous recipients of allogeneic hematopoietic stem cell transplantation (AHCT) are excluded if they are <100 days' post-transplant, have evidence of active graft-versushost-disease (GVHD) of any grade, or are currently on immunosuppression.  
11. Previous CAR-T cell therapy directed at either CD19 or CD20 within 100 days of planned CAR-20/19-T cell infusion (does not include re-enrollment)
a. Patients with prior CAR-T treatment against CD19 or CD20 must have repeat biopsy confirming a minimum of 5% CD19 or CD20 positivity by immunohistochemistry or flow cytometry 12. Anti-CD20 antibody treatment within 4 weeks of cell infusion 13. Anti-CD19 antibody treatment within 4 weeks of cell infusion 14. Cytotoxic chemotherapy other than lymphodepletion within 14 days of CAR-T cell infusion 15. Cytotoxic chemotherapy treatment within 14 days or steroid treatment (other than replacement dose steroids) within 7 days prior to apheresis collection for CAR-T cells 16. Patients post solid organ transplant who develop high grade lymphomas or leukemias 17. Concurrent active malignancy other than basal or squamous cell carcinomas of the skin
figure 1 study schema
4.4 special criteria for regarding

Female subjects of reproductive potential (women who have reached menarche or women who have not been post-menopausal for at least 24 consecutive months, i.e., who have had menses within the preceding 24 months, or have not undergone a sterilization procedure [hysterectomy or bilateral oophorectomy]) must have a negative serum or urine pregnancy test performed as part of eligibility criteria and again within 48 hours of initiation of lymphodepleting chemotherapy. Due to the high-risk level of this study, while enrolled, all subjects must agree not to participate in a conception process (e.g., active attempt to become pregnant or to impregnate, sperm donation, in vitro fertilization). Additionally, if participating in sexual activity that could lead to pregnancy, the study subject must agree to use reliable and double barrier methods of contraception during the follow-up period of the protocol.  
Acceptable birth control includes a combination of two of the following methods:
- Condoms* (male or female) with or without a spermicidal agent.  
- Diaphragm or cervical cap with spermicide - Intrauterine device (IUD)
- Hormonal-based contraception
Subjects who are not of reproductive potential (women who are premenarche or have been post-menopausal for at least 24 consecutive months or have undergone hysterectomy tubal ligation, salpingectomy, and/or bilateral oophorectomy or men who have documented azoospermia) are eligible without requiring the use of contraception.
figure 1 study schema
4.5 subject recruitment and screening

Subjects will be identified through the clinical practices of the investigator or sub-investigators and through referrals from outside hospitals and physicians. No direct-to-patient advertising will be performed.  
To be eligible, the subjects must have an adequate number of T cells (CD3+ ≥50/mm3) for CAR20/19-T cell line production, as determined from a peripheral blood sample before being considerable as eligible to undergo apheresis collection. The purpose of this screening procedure is to exclude subjects from participation who would otherwise undergo a futile apheresis and restaging. We will additionally determine the % of CD4 and CD8 in circulation to ensure the CAR-20/19-T line contains both helper and effector T cells.
figure 1 study schema
5 study entry and withdrawal

The study-specific assessments are detailed in this section and outlined in Table 1. Screening assessments must be performed within 8 weeks prior to enrollment. Any results falling outside of the reference ranges may be repeated at the investigator's discretion. All on-study visit procedures are allowed a window as noted in the study calendar. Treatment or visit delays for public holidays or weather conditions do not constitute a protocol violation.  
A written, signed informed consent form (ICF) and a Health Insurance Portability and Accountability Act (HIPAA) authorization must be obtained before any study-specific assessments are initiated. A signed ICF copy will be given to the subject and a copy will be filed in the medical record. The original will be kept on file with the study records.  
All patients who are consented will be registered in OnCore®, the MCW Cancer Center Clinical Trial Management System. The system is password protected and meets HIPAA requirements.
figure 1 study schema
registration process

Subjects will be identified through the clinical practices of the investigator or sub-investigators and through referrals from outside hospitals and physicians.  
To be eligible, patients need a diagnosis of CLL/SLL or B-cell NHL as specified in Section 4.  
CD19 or CD20 expression should be confirmed by IHC or flow cytometry (>5% expression of either CD19 or CD20) or MFC on malignant tissue, if it has not previously been documented. In the Phase 1 portion, 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. (e.g. 28 days between patient 1 and patient 2, and 28 days *between patient 2 and patient 3).*
For the Phase 1b portion, please see section 6.6 for details on dosing. The PI on the study, Dr.  
Nirav Shah, should be contacted regarding any questions regarding subject eligibility. Contact information for the PI is listed on the front page of the protocol.
figure 1 study schema
5.2 pretreatment period

Screening Assessments The screening procedures and assessments must be completed within ≤8 weeks prior to CAR20/19-T cells infusion (except as noted otherwise). For patients who fail to get their CAR-T cells within that time frame the studies below will have to be reported unless noted otherwise.  
Eligible subjects who have signed an informed consent and have adequate pre-screening evaluation will undergo a routine staging workup including:
- History and Physical Examination including height and weight, Performance Status Assessment (Karnofsky score), concomitant medications, and review of adverse events.  
- Vitals: Blood Pressure, Temperature, Respiratory rate, Pulse & Pulse Ox. - Radiologic imaging:
o Diagnostic PET-CT scans or CT of the Neck, Chest, Abdomen, and Pelvis; If >8 weeks' elapses between screening assessment and planned CAR-20/19-T cells infusion, scans will be required to be repeated.  
- Complete Blood Count and 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 - Ferritin, ESR, CRP
- Lipid Panel
- Beta-2-Microgloublin
- Creatinine Clearance
- Coagulation Assessments: PT/INR, PTT, Fibrinogen, and D-Dimer.  
- Thyroid Function Tests: TSH, Free T4
- Bone Marrow Transplant Markers/Apheresis Screening (Absolute numbers of CD3+ T
cells, CD4+ T cells and CD8+ T cells should be assessed before scheduling for apheresis collection to determine protocol eligibility. Minimum of ≥50 CD3+ T cells/mm3 required, with a mixture of both CD4+ and CD8+ T cells).  
- Quantitative Immunoglobulins (IgM, IgG, IgA) - Autoimmune Screen: ANA - Urinalysis - Serum or urine pregnancy test for females of child bearing potential - Infectious Disease Markers: HIV, Hepatitis B surface antigen, and HCV antibody within 8 weeks prior to CAR-20/19-T cells infusion. If the HCV antibody is positive, a screening HCV RNA by any RT-PCR or bDNA assay must be performed. Patients with HIV or Hep B positivity will be excluded from the study. *(Does not need to be repeated)*
- Bone marrow and aspirate evaluation within 8 weeks of CAR-20/19-T cells infusion
- Baseline cardiac ECHO/MUGA (Does not need to be repeated if no new cardiac symptoms)
- Electrocardiogram (ECG)
- Lumbar puncture as specified in Table 1 and inclusion criteria (Does not need to be repeated if no active CNS symptoms)
- MRI Brain (Only in patients with history of CNS disease or signs/symptoms of active CNS disease)
- Baseline neurological assessment
figure 1 study schema
5.3 enrollment

Documents required:
Signed informed consent forms and eligibility documentation Assignment of subject sequence number.
figure 1 study schema
5.4 study procedures related to

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
Apheresis T-cell repertoire diversity testing from apheresis sample Quality of Life Studies Tryptophan Metabolites PaxGene RNA gene expression
figure 1 study schema
5.4.2 mononuclear cell apheresis

A 2-5 blood volume apheresis will be targeted if tolerated by the patient, to be collected at Froedtert Hospital, as appropriate for CAR-20/19-T cell production. From a single apheresis, the intention is to harvest 1.0-5.0 x 109 MNC cells total to manufacture CAR-T cells.  
Baseline blood leukocytes for FDA requirements and for research are also obtained and cryopreserved.
figure 1 study schema
5.4.3 study procedures week -1

Patients will be seen 2-4 days prior to lymphodepleting chemotherapy.  
- Physical Examination including weight, concomitant medications, and review of adverse events.  
- Vital signs: Blood Pressure, Temperature, Respiratory rate, Pulse & Pulse Ox. - Urine or serum pregnancy test in females of childbearing potential
- Viral Respiratory NAAT will be performed in patients with signs/symptoms concerning for viral infection (cough, cold). In the setting of a positive result, initiation of LDP
chemotherapy will be delayed until resolution of symptoms
- Urinalysis
- Lumbar Puncture: For high risk patients with concern for CNS involvement, at the discretion of the PI, a repeat LP may be done prior to lymphodepleting chemotherapy
- If >8 weeks' elapse from staging bone marrow biopsy and aspirate, a repeat biopsy will be indicated
- Confirm patient has central line access
- Radiologic imaging:
o Diagnostic PET-CT scans or CT of the Neck, Chest, Abdomen, and Pelvis; If >8 weeks' elapses between screening assessment and planned CAR-20/19-T cells infusion, scans will be required to be repeated.
figure 1 study schema
5.4.4 start of lymphodepletion chemotherapy

Patients will then be seen again the day of initiation of Lymphodepletion chemotherapy. The following procedures must be completed prior to initiation of lymphodepleting chemotherapy  
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 T-cell repertoire diversity testing from CAR-T sample
A chemotherapy cycle (3 days of chemotherapy with a day of rest) is planned for all patients to start within approximately one week of CAR-20/19-T cell infusion. Patients will receive a combination of fludarabine/cyclophosphamide lymphodepletion. These chemotherapy agents are all commercially available and will be infused per institutional standards.  
Antimicrobial prophylaxis and TLS prophylaxis is outlined in **Appendix E.**
The chemotherapy will be planned so that it completes BEFORE the infusion of the first dose of CAR-20/19-T cells. The purpose of the chemotherapy is to induce lymphopenia to facilitate expansion of infused T cells. Although the chemotherapy is not investigational, patients will be required to receive it at Froedtert Hospital (FH) and/or the Medical College of Wisconsin Cancer Center. Patients will be started on allopurinol for tumor lysis prevention.
figure 1 study schema
5.4.5 car-20/19-t cell infusion (+

Infusion begins 1-7 days after the completion of chemotherapy and will be administered over two days (Day 0 and Day +1) in the Phase 1 portion. In the phase 1b portion cells will be administered as a single dose on Day 0. Patients can receive cells fresh, if appropriate or after cryopreservation for up to 6 weeks post-harvest. Patients will be either admitted to FH
under the care of the hematology/oncology service for the cell infusion or seen daily in the FH Day Hospital for infusion from Days 0-7. Details of the infusion and hospitalization plan can be found in the treatment plan section 6. On these days, the following evaluations will occur prior to CAR-20/19-T cells infusion unless stated otherwise:  
- 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 - Ferritin/ESR/CRP - Bone Marrow Transplant Markers (Day 0 only)
- Quantitative Immunoglobulins (IgM, IgG, IgA) only on Day 0 prior to infusion - Electrocardiogram (ECG)
- Coagulation Assessments: PT/INR, PTT, Fibrinogen, and D-Dimer. - Research Studies o T-Cell Persistent Studies: Baseline sample to be drawn prior to CAR-20/19-T
cells infusion on Day 0 only.  
o Cytokine Studies: At minimum, will include IFN-ϒ, IL10, IL6 and TNFα o Q-PCR for Integrated Vector DNA baseline sample on Day 0 prior to infusion o Detectable RCL by Q-PCR for VSV-G baseline sample on Day 0 prior to infusion